U.S. patent application number 12/622055 was filed with the patent office on 2010-03-25 for cyclic natriuretic peptide constructs.
This patent application is currently assigned to PALATIN TECHNOLOGIES, INC.. Invention is credited to Margarita Bastos, Hui-Zhi Cai, Shubh D. Sharma, Wei Yang.
Application Number | 20100075895 12/622055 |
Document ID | / |
Family ID | 38564246 |
Filed Date | 2010-03-25 |
United States Patent
Application |
20100075895 |
Kind Code |
A1 |
Sharma; Shubh D. ; et
al. |
March 25, 2010 |
Cyclic Natriuretic Peptide Constructs
Abstract
Cyclic constructs with an N-terminus and a C-terminus which bind
to a natriuretic peptide receptor and include a plurality of amino
acid residues, at least one amino acid surrogate of formula I:
##STR00001## where R, R', Q, Y, W, Z, J, x and n are as defined in
the specification, and optionally at least one prosthetic group,
pharmaceutical compositions including such cyclic constructs, and
methods of treating congestive heart failure or other conditions,
syndromes or diseases for which induction of anti-hypertensive,
cardiovascular, renal or endocrine effects are desired.
Inventors: |
Sharma; Shubh D.; (Cranbury,
NJ) ; Bastos; Margarita; (Plainsboro, NJ) ;
Yang; Wei; (Edison, NJ) ; Cai; Hui-Zhi; (East
Brunswick, NJ) |
Correspondence
Address: |
PALATIN TECHNOLOGIES, INC.
4-C CEDAR BROOK DRIVE, CEDAR BROOK CORPORATE CENTER
CRANBURY
NJ
08512
US
|
Assignee: |
PALATIN TECHNOLOGIES, INC.
Cranbury
NJ
|
Family ID: |
38564246 |
Appl. No.: |
12/622055 |
Filed: |
November 19, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11694260 |
Mar 30, 2007 |
7622440 |
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12622055 |
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60743960 |
Mar 30, 2006 |
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60743961 |
Mar 30, 2006 |
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Current U.S.
Class: |
514/1.1 ;
530/317 |
Current CPC
Class: |
C07K 14/58 20130101 |
Class at
Publication: |
514/9 ;
530/317 |
International
Class: |
A61K 38/12 20060101
A61K038/12; C07K 7/50 20060101 C07K007/50 |
Claims
1. A cyclic peptide construct with an N-terminus and a C-terminus
comprising from eleven to thirteen amino acid residues with an
amino acid surrogate at the C-terminus position of the following
formula: wherein ##STR00411## Y is CH.sub.2 or C.dbd.O; W is
CH.sub.2, NH or NR''; Z is H or CH.sub.3; J is --H, --OH,
--C(.dbd.O)--OH, --C(.dbd.O)--NH.sub.2, or a C-terminus capping
group; one of R.sup.a and R.sup.b is H and the other of R.sup.a and
R.sup.b is --(CH.sub.2).sub.y--R''; R'' is: --NH.sub.2,
--NH--C(.dbd.NH)--NH.sub.2, --NH--(CH.sub.2).sub.y--NH.sub.2,
--NH--C(.dbd.O)--NH.sub.2, --C(.dbd.O)--NH.sub.2,
--C(.dbd.O)--NH--CH.sub.3,
--C(.dbd.O)--NH--(CH.sub.2).sub.y--NH.sub.2,
--NH--C(.dbd.NH)--NH-Me, --NH--C(.dbd.NH)--NH-Et,
--NH--C(.dbd.NH)--NH--Pr, --NH--C(.dbd.NH)--NH--Pr-i,
--NH--C(.dbd.O)--CH.sub.3, --NH--C(.dbd.O)--CH.sub.2--CH.sub.3,
--NH--C(.dbd.O)--CH--(CH.sub.3).sub.2,
--NH--C(.dbd.O)--O--CH.sub.3,
--NH--C(.dbd.O)--O--CH.sub.2--CH.sub.3,
--NH--C(.dbd.O)--O--C--(CH.sub.3).sub.3,
--NH--C(.dbd.O)--NH--CH.sub.3,
--NH--C(.dbd.N--C(.dbd.O)--O--C--(CH.sub.3).sub.3)--NH--C(.dbd.O)--O--C---
(CH.sub.3).sub.3,
--N(C(.dbd.O)--O--C--(CH.sub.3).sub.3)--C(.dbd.NH)--NH--C(.dbd.O)--O--C---
(CH.sub.3).sub.3, ##STR00412## R''' is an acyl, a C.sub.1 to
C.sub.17 linear or branched alkyl chain, a C.sub.2 to C.sub.19
linear or branched alkyl acyl chain, a C.sub.i to C.sub.17 linear
or branched omega amino aliphatic, or a C.sub.i to C.sub.17 linear
or branched omega amino aliphatic acyl; x is 1 or 2; y is 1 to 5; n
is 0, 1 or 2; and the carbon atoms marked with an asterisk can have
any stereochemical configuration; with the amino acid surrogate at
the C-terminus position being covalently bonded to the immediately
adjacent amino acid residue.
2. The cyclic peptide construct of claim 1, wherein the C-terminus
capping group is: --(CH.sub.2).sub.m--OH,
--C(.dbd.O)--(CH.sub.2).sub.m--N(v.sub.1)(v.sub.2),
--C(.dbd.O)--O--(CH.sub.2).sub.m--CH.sub.3,
--O--(CH.sub.2).sub.m--CH.sub.3,
--O--(CH.sub.2).sub.m--N(v.sub.1)(v.sub.2),
--O--(CH.sub.2).sub.m--OH,
--C(.dbd.O)--NH--(CH.sub.2).sub.m--S(v.sub.1),
--C(.dbd.O)--NH--(CH.sub.2).sub.m--CH.sub.3,
--C(.dbd.O)--NH--(CH.sub.2).sub.m--N(v.sub.1)(v.sub.2),
--C(.dbd.O)--N--((CH.sub.2).sub.m--N(v.sub.1)(v.sub.2)).sub.2,
--C(.dbd.O)--NH--CH(--C(.dbd.O)--OH)--(CH.sub.2).sub.m--N(v.sub.1)(v.sub.-
2),
--C(.dbd.O)--NH--(CH.sub.2).sub.m--NH--C(.dbd.O)--CH(N(v.sub.1)(v.sub.-
2))((CH.sub.2).sub.m--N(v.sub.1)(v.sub.2)), or
--C(.dbd.O)--NH--CH(--C(.dbd.O)--N(v.sub.1)(v.sub.2))--(CH.sub.2).sub.m---
N(v.sub.1)(v.sub.2), including all (R) or (S) configurations of the
foregoing; v.sub.1 and v.sub.2 are each independently H or a
C.sub.1 to C.sub.17 linear or branched alkyl chain; and m is in
each instance independently 0 to 17.
3. The cyclic peptide construct of claim 1, comprising at least one
further amino acid surrogate at any position other than the
C-terminus position of the following formula: ##STR00413## wherein
Q is a bond unless the surrogate is at the N-terminus position of
the construct, in which case Q is --H or an amine capping group;
and R.sup.c and R.sup.d are each independently H or a natural or
unnatural amino acid side chain moiety or derivative of an amino
acid side chain moiety.
4. The cyclic peptide construct of claim 3, wherein the at least
one further amino acid surrogate is at the N-terminus position of
the construct and Q is an amine capping group of the formula:
--(CH.sub.2).sub.m--N(v.sub.3)(v.sub.4),
--(CH.sub.2).sub.m--CH.sub.3, --(CH.sub.2).sub.m--O(v.sub.3),
--(CH.sub.2).sub.m--C(.dbd.O)-(v.sub.3),
--(CH.sub.2).sub.m--C(.dbd.O)--O-(v.sub.3),
--(CH.sub.2).sub.m--S(v.sub.3),
--C(.dbd.O)--(CH.sub.2).sub.m--CH.sub.3,
--C(.dbd.O)--(CH.sub.2).sub.m--N(v.sub.3)(v.sub.4),
--C(.dbd.O)--(CH.sub.2).sub.m--C(.dbd.O)-(v.sub.3),
--C(.dbd.O)--(CH.sub.2).sub.m--O(v.sub.3), or
--C(.dbd.O)--(CH.sub.2).sub.m--S(v.sub.3); v.sub.3 and v.sub.4 are
each independently H, a C.sub.1 to C.sub.17 linear or branched
alkyl chain or a C.sub.2 to C.sub.19 linear or branched alkyl acyl
chain, on the proviso that if one of v.sub.3 or v.sub.4 is an alkyl
acyl chain, then the other of v.sub.3 or v.sub.4 is H; and m is 0
to 17.
5. The cyclic peptide construct of claim 1, further comprising an
N-terminus acyl comprising a C.sub.2 to C.sub.18 linear alkyl, a
C.sub.3 to C.sub.17 branched alkyl, a C.sub.2 to C.sub.18 linear
alkenyl or alkynyl or a C.sub.3 to C.sub.18 branched alkenyl or
alkynyl.
6. The cyclic peptide construct of claim 5 wherein the N-terminus
acyl is -C(.dbd.O)--(CH.sub.2).sub.p--CH.sub.3 where p is 1 to
18.
7. The cyclic peptide construct of claim 6 of the formula:
##STR00414##
8. The cyclic peptide construct of claim 1, wherein the amino acid
residues comprise natural or unnatural .alpha.-amino acids,
.beta.-amino acids, N-substituted amino acids, or .alpha.,
.alpha.-disubstituted amino acids, including all (R) or (S)
configurations of any of the foregoing, or any combination of the
foregoing.
9. The cyclic peptide construct of claim 1, wherein at least two of
the amino acid residues are joined by a non-peptide bond.
10. The cyclic peptide construct of claim 9, wherein the at least
one non-peptide bond is --CH.sub.2--NH--, --CH.sub.2--S--,
--CH.sub.2--O--, or --C(.dbd.O)--CH.sub.2--, an isostere of any of
the foregoing, or --CH.sub.2--CH.sub.2-- or --CH.dbd.CH--.
11. The cyclic peptide construct of claim 1, wherein the cyclic
peptide construct binds to a natriuretic peptide receptor selected
from the group consisting of a receptor for ANP, BNP, CNP, sCP,
DNP, TNP-a, TNP-b or TNP-c.
12. The cyclic peptide construct of claim 11, wherein the cyclic
peptide construct exhibits, upon administration to a mammal, one or
more advantages relative to the corresponding amino acid sequence
not comprising an amino acid surrogate, the advantages selected
from the group consisting of increased resistance to enzymatic
degradation, increased circulation half life, increased
bioavailability, increased efficacy, and prolonged duration of
effect.
13. The cyclic peptide construct of claim 1, wherein Y is C.dbd.O,
W is NH, Z is H, x is 1 and n is 0.
14. The cyclic peptide construct of claim 1, further comprising at
least one prosthetic group covalently bonded to a reactive group in
a side chain or terminal group of at least one of the amino acid
residues or to a carboxyl group, amine group or reactive group in a
C-terminus capping group of the amino acid surrogate.
15. The cyclic peptide construct of claim 14, wherein the
prosthetic group comprises at least one polymeric sequence
comprising repeat units including carbon and hydrogen atoms.
16. The cyclic peptide construct of claim 15, wherein the polymeric
sequence is poly(alkylene oxide), poly(vinyl pyrrolidone),
poly(vinyl alcohol), polyoxazoline or poly(acryloylmorpholine).
17. The cyclic peptide construct of claim 16, wherein the
poly(alkylene oxide) is poly(ethylene glycol) (PEG).
18. The cyclic peptide construct of claim 17, wherein the
prosthetic group comprising PEG is derivatized with a linking
group.
19. A pharmaceutical composition, comprising a cyclic peptide
construct of claim 1 or a pharmaceutically acceptable salt of a
cyclic peptide construct of claim 1 and a pharmaceutically
acceptable carrier.
20. A pharmaceutical composition, comprising a cyclic peptide
construct of claim 7 or a pharmaceutically acceptable salt of a
cyclic peptide construct of claim 7 and a pharmaceutically
acceptable carrier.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application to U.S. Ser.
No. 11/694,260 entitled "Cyclic Natriuretic Peptide Constructs",
filed on Mar. 30, 2007, now U.S. Pat. No. 7,622,440, issued on Nov.
24, 2009.
[0002] U.S. Ser. No. 11/694,260 claims priority to and the benefit
of the filing of U.S. Provisional Patent Application Ser. No.
60/743,960 entitled "Cyclic Natriuretic Peptide Constructs", filed
on Mar. 30, 2006, and of U.S. Provisional Patent Application Ser.
No. 60/743,961 entitled "Cyclic Natriuretic Peptide Constructs with
Prosthetic Groups", filed on Mar. 30, 2006, and the specification
and claims thereof of each are incorporated herein by
reference.
[0003] A related application entitled "Amino Acid Surrogates for
Peptidic Constructs" was filed on Mar. 30, 2007, U.S. patent
application Ser. No. 11/694,181, Attorney Docket No. 0307-043, and
the specification and claims thereof are incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0004] 1. Field of the Invention (Technical Field)
[0005] The present invention relates to cyclic natriuretic peptide
constructs which include a plurality of amino acid residues, one or
more ring-constrained amino acid surrogates and optionally one or
more prosthetic groups, which constructs bind a natriuretic peptide
receptor and may be employed for therapeutic purposes.
[0006] 2. Background Art
[0007] The natriuretic peptide system has been extensively explored
since the identification of the human atrial natriuretic peptide
(ANP) sequence and gene structure in 1984. ANP is sometimes also
called "ANF", or atrial natriuretic factor. ANP is part of the
natriuretic peptide system, which in humans involves an ANP gene,
which through differences in post-translational processing results
in both ANP and urodilatin, a gene which produces BNP, or brain
natriuretic peptide, and a gene which produces CNP, or c-type
natriuretic peptide. ANP, urodilatin, BNP and CNP are each ring
structures, with a 17 amino acid loop formed by a cysteine-cysteine
disulfide linkage. The amino acid sequence and structure of human
ANP (hANP) is shown in FIG. 1. ANP, urodilatin, BNP and CNP are
closely related, differing by some five or six amino acids within
the ring, though the N- and C-terminal tails are substantially
different.
[0008] There are three known natriuretic peptide receptors called
natriuretic peptide receptors A, B and C (NPRA, NPRB and NPRC).
NPRA and NPRB are linked to guanylyl cyclases, while NPRC is a
G-protein linked clearance receptor. ANP, BNP and CNP are the
primary endogenous mammalian natriuretic peptides identified to
date. However, there are a number of non-mammalian natriuretic
peptides that have been identified and may have therapeutic
application in mammals. These include salmon natriuretic or cardiac
peptide (sCP), ventricular natriuretic peptide (VNP), a cardiac
natriuretic peptide identified in eels and a variety of fish,
dendroaspis natriuretic peptide (DNP), a natriuretic peptide
identified in mamba snake venom, and three natriuretic-like
peptides (TNP-a, TNP-b, and TNP-c) isolated from taipan snake
venom. See generally Tervonen V, Ruskoaho H, Lecklin T, Ilves M,
Vuolteenaho O, Salmon cardiac natriuretic peptide is a
volume-regulating hormone. Am. J. Physiol. Endocrinol. Metab.
283:E353-61 (2002); Takei Y, Fukuzawa A, Itahara Y, Watanabe T X,
Yoshizawa Kumagaye K, Nakajima K, Yasuda A, Smith M P, Duff D W,
Olson K R. A new natriuretic peptide isolated from cardiac atria of
trout, Oncorhynchus mykiss. FEBS Lett. 414:377-80 (1997); Schweitz
H, Vigne P, Moinier D, Frelin C, Lazdunski M. A new member of the
natriuretic peptide family is present in the venom of the green
mamba (Dendroaspis angusticeps). J. Biol. Chem. 267:13928-32
(1992); Lisy 0, Jougasaki M, Heublein D M, Schirger J A, Chen H H,
Wennberg P W, Burnett J C. Renal actions of synthetic dendroaspis
natriuretic peptide. Kidney Int. 56:502-8 (1999); and Fry B G,
Wickramaratana J C, Lemme S, Beuve A, Garbers D, Hodgson W C,
Alewood P. Novel natriuretic peptides from the venom of the inland
(Oxyuranus microlepidotus): isolation, chemical and biological
characterisation. Biochem. Biophys. Res. Comm. 327:1011-1015
(2005).
[0009] ANP is endogenously secreted predominately in response to
increased atrial pressure, but other factors, including cytokine
receptor stimulation, may contribute to endogenous secretion. Once
released, ANP is a hormonal regulator of blood pressure, sodium and
fluid homeostasis, providing vasorelaxant effects, affecting
cardiovascular remodeling, and the like. Thus ANP, including
endogenous ANP, is effective in congestive heart failure and other
cardiovascular disease, in part by providing a defense against a
chronically activated renin-angiotensin-aldosterone system.
Circulating ANP is rapidly removed from the circulation by two
mechanisms, binding to a natriuretic peptide receptor and enzymatic
degradation.
[0010] Human ANP is also referred to as wild-type human ANP, hANP,
ANP(1-28) and ANP(99-126) (the later referring to the relevant
sequence within proANP(1-126), which is normally cleaved at
Arg.sup.98-Ser.sup.99 in the C-terminal region during secretion).
Hereafter human ANP is sometimes referred to as "hANP."
[0011] In general, natriuretic peptides and variants thereof are
believed to have utility in the treatment of congestive heart
failure, renal hypertension, acute kidney failure and related
conditions, as well as any condition, disease or syndrome for which
a diuretic, natriuretic and/or vasodilatory response would have a
therapeutic or preventative effect. One review article describing
natriuretic peptides, including ANP, and use of the natriuretic
peptide system in heart failure is Schmitt M., Cockcroft J. R., and
Frenneaux M. P. Modulation of the natriuretic peptide system in
heart failure: from bench to bedside? Clinical Science 105:141-160
(2003).
[0012] A large number of ANP mimetics and variations have been
made, some of which are substantially reduced in size from ANP. On
ANP version that is reduced in size yet is biologically active is
the 15-mer disulfide cyclic peptide
H-Met-cyclo(Cys-His-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Ser-Cys)-Tyr-Arg-NH.s-
ub.2 (SEQ ID NO:1) as described in Li B, Tom J Y, Oare D, Yen R,
Fairbrother W J, Wells J A, Cunningham B C. Minimization of a
polypeptide hormone. Science 270:1657-60 (1995). This 15-mer
peptide is commonly referred to as "mini-ANP".
[0013] A number of patents and patent applications have been filed
on different synthetic mimics of natriuretic peptides, asserted to
be superior to wild-type natriuretic peptides based on one or more
factors. These include the constructs disclosed in the following
U.S. Pat. Nos. 4,496,544; 4,609,725; 4,656,158; 4,673,732;
4,716,147; 4,757,048; 4,764,504; 4,804,650; 4,816,443; 4,824,937;
4,861,755; 4,904,763; 4,935,492; 4,952,561; 5,047,397; 5,057,495;
5,057,603; 5,091,366; 5,095,004; 5,106,834; 5,114,923; 5,159,061;
5,204,328; 5,212,286; 5,352,587; 5,376,635; 5,418,219; 5,665,704;
5,846,932; 5,583,108; 5,965,533; 6,028,055; 6,083,982; 6,124,430;
6,150,402; 6,407,211; 6,525,022; 6,586,396 and 6,818,619; and in
the following U.S. Patent Application Publications: 2004/0002458;
2004/0063630; 2004/0077537; 2005/0113286; 2005/0176641;
2006/0030004. In addition, various non-U.S. patents and patent
applications disclose constructs, including: WO 85/04870; WO
85/04872; WO 88/03537; WO 88/06596; WO 89/10935; WO 89/05654; WO
90/01940; WO 90/14362; WO 92/06998; WO 95/13296; WO 99/08510; WO
99/12576; WO 01/016295; WO 2004/047871; WO 2005/072055; EPO 0 291
999; EPO 0 323 740; EPO 0 341 603; EPO 0 350 318; EPO 0 356 124;
EPO 0 385 476; EPO 0 497 368; and EPO 0 542 863. Chimeric
natriuretic peptides, such as a peptide call "vasonatrin peptide"
and described as a chimera of ANP and CNP, are described, as in
U.S. Pat. No. 5,583,108, or in U.S. Pat. Nos. 6,407,211 and
6,818,619, disclosing chimeric peptides of dendroaspis. The
teachings of each of the foregoing patents and patent applications
are incorporated by reference as if set forth in full.
[0014] There is one natriuretic peptide product approved by the
Food and Drug Administration in the United States, sold under the
generic name nestiritide and the tradename Natrecor.RTM. (Scios
Inc.). This is a human B-type natriuretic peptide manufactured from
E. coli using recombinant DNA technology. This product is approved
only for intravenous infusion for treatment of patients with
actutely decompensated congestive heart failure who have dyspnea at
rest or with minimal activity. While effective, the
pharmacokinetics and half-life of nestiritide are such that the
product can only be employed by intravenous infusion, which limits
use of the drug to a hospital or skilled medical center
setting.
[0015] Notwithstanding the large number of compounds that have been
developed, virtually none are commercialized or in active clinical
development. There is a substantial need for products with improved
characteristics, including improved potency, half-life, modes of
administration, bioavailability or prolonged duration of effect,
which products are effective for one or more therapeutic
indications, and which preferably may be administered on an
out-patient basis.
BRIEF SUMMARY OF THE INVENTION
[0016] In one aspect the invention provides a cyclic construct
which binds to a receptor for a natriuretic peptide, including but
not limited to a receptor for ANP, BNP, CNP, sCP, DNP, TNP-a, TNP-b
or TNP-c, wherein such construct includes a plurality of amino acid
residues, at least one amino acid surrogate of the general formula
I:
##STR00002##
where R and R' are each independently H or a natural or unnatural
amino acid side chain moiety or derivative of an amino acid side
chain moiety; x is 1 or 2; Y is CH.sub.2 or C.dbd.O; W is CH.sub.2,
NH or NR'''; Z is H or CH.sub.3; n is 0, 1 or 2; J is --C(.dbd.O)--
unless the surrogate is at the C-terminus position of the
construct, in which case J is --H, --OH, --C(.dbd.O)--OH,
--C(.dbd.O)--NH.sub.2 or a C-terminus capping group; Q is a bond
unless the surrogate is at the N-terminus position of the
construct, in which case Q is --H or an amine capping group; R'''
is an acyl, a C.sub.1 to C.sub.17 linear or branched alkyl chain, a
C.sub.2 to C.sub.19 linear or branched alkyl acyl chain, a C.sub.1
to C.sub.17 linear or branched omega amino aliphatic, or a C.sub.1
to C.sub.17 linear or branched omega amino aliphatic acyl;
optionally at least one prosthetic group covalently bonded to a
reactive group in a side chain of at least one of the amino acid
residues, to an amine capping group where the surrogate is at the
N-terminus position of the construct, or to a C-terminus capping
group where the surrogate is at the C-terminus position of the
construct; and the carbon atoms marked with an asterisk can have
any stereochemical configuration. The construct is a cyclic
construct, cyclized by a bond between side chains of two amino acid
residues, between an amino acid residue side chain and an R or R'
group of an amino acid surrogate, between R or R' groups of two
amino acid surrogates, between a terminal group of the construct
and an amino acid residue side chain, or between a terminal group
of the construct and an R or R' group of an amino acid surrogate.
Preferable the two amino acid residues forming a bond between the
side chains thereof are separated by between about eight and ten
amino acid residues and optionally zero, one or two amino acid
surrogates. The plurality of amino acid residues may include any
amino acid residue selected from the group consisting of natural or
unnatural .alpha.-amino acids, .beta.-amino acids, .alpha.,
.alpha.-disubstituted amino acids and N-substituted amino acids,
including all (R) or (S) configurations of any of the
foregoing.
[0017] The prosthetic group(s) may include polymeric groups
comprising repeat units including one or more carbon and hydrogen
atoms, and optionally other atoms, including oxygen. Such polymeric
groups are preferably water-soluble polymers, and are preferably
poly(alkylene oxide), poly(vinyl pyrrolidone), poly(vinyl alcohol),
polyoxazoline or poly(acryloylmorpholine). A preferred
poly(alkylene oxide) is poly(ethylene glycol) (PEG), optionally
derivatized with a linking group.
[0018] In one aspect, J is a C-terminus capping group selected from
[0019] --(CH.sub.2).sub.m--OH, [0020]
--C(.dbd.O)--(CH.sub.2).sub.m--N(v.sub.1)(v.sub.2), [0021]
--C(.dbd.O)--O--(CH.sub.2).sub.m--CH.sub.3, [0022]
--O--(CH.sub.2).sub.m--CH.sub.3, [0023]
--O--(CH.sub.2).sub.m--N(v.sub.1)(v.sub.2), [0024]
--O--(CH.sub.2).sub.m--OH, [0025]
--C(.dbd.O)--NH--(CH.sub.2).sub.m--S(v.sub.1), [0026]
--C(.dbd.O)--NH--(CH.sub.2).sub.m--CH.sub.3, [0027]
--C(.dbd.O)--NH--(CH.sub.2).sub.m--N(v.sub.1)(v.sub.2), [0028]
--C(.dbd.O)--N--((CH.sub.2).sub.m--N(v.sub.1)(v.sub.2)).sub.2,
[0029]
--C(.dbd.O)--NH--CH(--C(.dbd.O)--OH)--(CH.sub.2).sub.m--N(v.sub.1)(v.sub.-
2), [0030]
--C(.dbd.O)--NH--(CH.sub.2).sub.m--NH--C(.dbd.O)--CH(N(v.sub.1)-
(v.sub.2))((CH.sub.2).sub.m--N(v.sub.1)(v.sub.2)), or [0031]
--C(.dbd.O)--NH--CH(--C(.dbd.O)--N(v.sub.1)(v.sub.2))--(CH.sub.2).sub.m---
N(v.sub.1)(v.sub.2); including all (R) or (S) configurations of the
foregoing, where v.sub.1 and v.sub.2 are each independently H or a
C.sub.1 to C.sub.17 linear or branched alkyl chain and m is in each
instance independently 0 to 17.
[0032] In another aspect where the amino acid surrogate is at the
C-terminus position of the construct, J is a C-terminus capping
group consisting of an omega amino aliphatic, terminal aryl or
aralkyl group or any single natural or unnatural .alpha.-amino
acid, .beta.-amino acid, .alpha., .alpha.-disubstituted amino acid
or N-substituted amino acid, including all (R) or (S)
configurations of an .alpha., .alpha.-disubstituted amino acid
where the substituents are different, optionally in combination
with a C-terminus capping group as defined above.
[0033] In another aspect, Q is an amine capping group selected from
[0034] --(CH.sub.2).sub.m--N(v.sub.3)(v.sub.4), [0035]
--(CH.sub.2).sub.m--CH.sub.3, [0036]
--(CH.sub.2).sub.m--O(v.sub.3), [0037]
--(CH.sub.2).sub.m--C(.dbd.O)-(v.sub.3), [0038]
--(CH.sub.2).sub.m--C(.dbd.O)--O-(v.sub.3), [0039]
--(CH.sub.2).sub.m--S(v.sub.3), [0040]
--C(.dbd.O)--(CH.sub.2).sub.m--CH.sub.3, [0041]
--C(.dbd.O)--(CH.sub.2).sub.m--N(v.sub.3)(v.sub.4), [0042]
--C(.dbd.O)--(CH.sub.2).sub.m--C(.dbd.O)-(v.sub.3), [0043]
--C(.dbd.O)--(CH.sub.2).sub.m--O(v.sub.3), or [0044]
--C(.dbd.O)--(CH.sub.2).sub.m--S(v.sub.3); where v.sub.3 and
v.sub.4 are each independently H, a C.sub.1 to C.sub.17 linear or
branched alkyl chain or a C.sub.2 to C.sub.19 linear or branched
alkyl acyl chain, on the proviso that if one of v.sub.3 or v.sub.4
is an alkyl acyl chain, then the other of v.sub.3 or v.sub.4 is H,
and m is 0 to 17.
[0045] In a related aspect, an amino acid surrogate of formula I is
at the C-terminus position of the construct, and at least one of R
and R' is a natural or unnatural amino acid side chain moiety or
derivative of an amino acid side chain moiety with a heteroatom
group comprising at least one nitrogen atom, and the remaining one
of R and R' is H or a natural or unnatural amino acid side chain
moiety or derivative of an amino acid side chain moiety.
[0046] In a related embodiment, the invention provides a construct
which binds to a receptor for a natriuretic peptide, including but
not limited to a receptor for ANP, BNP, CNP, sCP, DNP, TNP-a, TNP-b
or TNP-c, wherein such construct includes a plurality of amino acid
residues and at least one amino acid surrogate located at any
position other than the C-terminus position or N-terminus position
and covalently bonded by two peptide bonds, and of formula II:
##STR00003##
where R and R' are each independently H or a natural or unnatural
amino acid side chain moiety or derivative of an amino acid side
chain moiety; x is 1 or 2; Y is CH.sub.2 or C.dbd.O; W is CH.sub.2,
NH or NR'''; Z is H or CH.sub.3; R''' is an acyl, a C.sub.1 to
C.sub.17 linear or branched alkyl chain, a C.sub.2 to C.sub.19
linear or branched alkyl acyl chain, a C.sub.1 to C.sub.17 linear
or branched omega amino aliphatic, or a C.sub.1 to C.sub.17 linear
or branched omega amino aliphatic acyl; n is 0, 1 or 2; the carbon
atoms marked with an asterisk can have any stereochemical
configuration; and the broken lines indicate the bond forming a
peptide bond.
[0047] Where the surrogate of formula I is at the C-terminus of the
construct, it is covalently bonded thereto by a single peptide
bond, such that the surrogate has the formula:
##STR00004##
where the broken line indicates the bond forming a peptide bond.
Where the surrogate is at the N-terminus of the construct it is
preferably of formula I, and is covalently bonded thereto by a
single bond peptide bond, such that the surrogate has the
formula:
##STR00005##
where the broken line indicates the bond forming a peptide bond.
However, where the surrogate is at other than at the N-terminus or
C-terminus of the construct, it is preferably of formula II and is
covalently bonded thereto by two peptide bonds.
[0048] In different embodiments of the invention, one amino acid
surrogate may be employed in a construct of the invention, two
amino acid surrogates may be employed in a construct of the
invention, or more than two amino acid surrogates may be employed
in a construct of the invention.
[0049] In another preferred embodiment, the invention provides a
construct wherein one or more peptide bonds between amino acid
residues are substituted with a non-peptide bond.
[0050] A primary object of the present invention is to provide
natriuretic receptor-specific constructs.
[0051] Another object of the present invention is to provide
natriuretic receptor-specific constructs wherein one or more amino
acid residues are substituted by a ring-constrained amino acid
surrogate.
[0052] Another object of the present invention is to provide a
natriuretic receptor-specific construct wherein the construct
exhibits, upon administration to a mammal, one or more advantages
relative to the corresponding amino acid sequence not comprising an
amino acid surrogate, the advantages selected from the group
consisting of increased resistance to enzymatic degradation,
increased circulation half life, increased bioavailability,
increased efficacy, prolonged duration of effect and combinations
of the foregoing.
[0053] Another object of the present invention is to provide a
natriuretic receptor-specific construct wherein the construct has
at least 10% of the maximal cGMP stimulating activity as the same
concentration of the corresponding amino acid sequence not
comprising an amino acid surrogate.
[0054] Another object of the present invention is to provide a
natriuretic receptor-specific construct wherein the construct has
at least 50% of the maximal cGMP stimulating activity as the same
concentration of the corresponding amino acid sequence not
comprising an amino acid surrogate.
[0055] Another object of the present invention is to provide a
natriuretic receptor-specific construct wherein the construct has
at least 100% of the maximal cGMP stimulating activity as the same
concentration of the corresponding amino acid sequence not
comprising an amino acid surrogate.
[0056] Another object of the present invention is to provide a
natriuretic receptor-specific construct wherein the construct has
more than 100% of the maximal cGMP stimulating activity as the same
concentration of the corresponding amino acid sequence not
comprising an amino acid surrogate.
[0057] Another object of the present invention is to provide a
natriuretic receptor-specific construct wherein the construct has
an equilibrium receptor binding affinity, determined by the Ki (nM)
value, no greater than two log orders higher than the Ki (nM) value
of the corresponding amino acid sequence not comprising an amino
acid surrogate.
[0058] Another object of the present invention is to provide a
natriuretic receptor-specific construct wherein the construct has
an equilibrium receptor binding affinity, determined by the Ki (nM)
value, no greater than three times higher than the Ki (nM) value of
the corresponding amino acid sequence not comprising an amino acid
surrogate.
[0059] Another object of the present invention is to provide a
natriuretic receptor-specific construct wherein the construct has
an equilibrium receptor binding affinity, determined by the Ki (nM)
value, equal to or less than than the Ki (nM) value of the
corresponding amino acid sequence not comprising an amino acid
surrogate.
[0060] Another object of the present invention is to provide a
natriuretic receptor-specific construct wherein the construct has
an equilibrium receptor binding affinity, determined by the Ki (nM)
value, less than the Ki (nM) value of the corresponding amino acid
sequence not comprising an amino acid surrogate.
[0061] Another object of the present invention is to provide a
natriuretic receptor-specific construct wherein the construct has a
receptor binding affinity with respect to a natriuretic peptide
receptor greater than the receptor binding affinity of the
corresponding amino acid sequence not comprising an amino acid
surrogate.
[0062] Another object of the present invention is to provide a
natriuretic receptor-specific construct wherein the construct has
biological efficacy, determined by decrease in blood pressure or
increase in urine output over time, at least as efficacious as or
more efficacious than than the same dose of the corresponding amino
acid sequence not comprising an amino acid surrogate.
[0063] Another object of the present invention is to provide a
natriuretic receptor-specific construct wherein the construct has
biological efficacy, determined by decrease in blood pressure or
increase in urine output over time, more efficacious than than the
same dose of the corresponding amino acid sequence not comprising
an amino acid surrogate.
[0064] Another object of the present invention is to provide a
natriuretic receptor-specific construct wherein the corresponding
amino acid sequence not comprising an amino acid surrogate has at
least about 60% homology with the sequence of a natriuretic
peptide.
[0065] Another object of the present invention is to provide a
natriuretic receptor-specific construct wherein the corresponding
amino acid sequence not comprising an amino acid surrogate has at
least about 80% homology with the sequence of a natriuretic
peptide.
[0066] Another object of the present invention is to provide a
natriuretic receptor-specific construct wherein the corresponding
amino acid sequence not comprising an amino acid surrogate has at
least about 60% homology with the sequence of a peptide that binds
to a receptor for ANP, BNP, CNP, sCP, DNP, TNP-a, TNP-b or
TNP-c.
[0067] Another object of the present invention is to provide a
natriuretic receptor-specific construct wherein the corresponding
amino acid sequence not comprising an amino acid surrogate has at
least about 80% homology with the sequence of a peptide that binds
to a receptor for ANP, BNP, CNP, sCP, DNP, TNP-a, TNP-b or
TNP-c.
[0068] Another object of the present invention is to provide a
natriuretic receptor-specific construct wherein the corresponding
amino acid sequence not comprising an amino acid surrogate has at
least about 60% homology with the sequence
H-Met-cyclo(Cys-His-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Ser-Cys)-Tyr-Arg-NH.s-
ub.2 (SEQ ID NO:1).
[0069] Another object of the present invention is to provide a
natriuretic receptor-specific construct wherein the corresponding
amino acid sequence not comprising an amino acid surrogate has at
least about 80% homology with the sequence
H-Met-cyclo(Cys-His-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Ser-Cys)-Tyr-Arg-NH.s-
ub.2 (SEQ ID NO:1).
[0070] Another object of the present invention is to provide a
natriuretic receptor-specific construct wherein the corresponding
amino acid sequence not comprising an amino acid surrogate has at
least about 60% homology with the sequence
H-Met-cyclo(Xaa-His-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Ser-Xaa)-Tyr-Arg-NH.s-
ub.2 (SEQ ID NO:2), where Xaa are each independently any amino acid
residue together forming a cyclic peptide.
[0071] Another object of the present invention is to provide a
natriuretic receptor-specific construct wherein the corresponding
amino acid sequence not comprising an amino acid surrogate has at
least about 80% homology with the sequence
H-Met-cyclo(Xaa-His-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Ser-Xaa)-Tyr-Arg-NH.s-
ub.2 (SEQ ID NO:2), where Xaa are each independently any amino acid
residue together forming a cyclic peptide.
[0072] Another object of the present invention is to provide a
natriuretic receptor-specific construct including a surrogate as
defined herein wherein the corresponding amino acid sequence not
comprising an amino acid surrogate is a peptide which binds to a
receptor for ANP.
[0073] Another object of the present invention is to provide a
natriuretic receptor-specific construct including a surrogate as
defined herein wherein the corresponding amino acid sequence not
comprising an amino acid surrogate is a peptide which binds to a
receptor for BNP.
[0074] Another object of the present invention is to provide
natriuretic receptor-specific constructs with greater
bioavailability and half-life than natural or recombinant forms of
ANP or BNP.
[0075] Another object of the present invention is to provide
natriuretic receptor-specific constructs which may be administered
to patients with congestive heart failure.
[0076] Another object of the present invention is to provide
natriuretic receptor-specific constructs which may be administered
by at least one route of administration in addition to intravenous
administration.
[0077] Another object of the present invention is to provide
natriuretic receptor-specific constructs which may be administered
to patients by subcutaneous or intramuscular injection.
[0078] Another object of the present invention is to provide
natriuretic receptor-specific constructs with increased resistance
to degradation but which have a significantly high binding affinity
to its receptor.
[0079] Another object of the present invention is to provide
natriuretic receptor-specific constructs in a sustained release
formulation.
[0080] Other objects, advantages and novel features, and further
scope of applicability of the present invention will be set forth
in part in the detailed description to follow, taken in conjunction
with the accompanying drawings, and in part will become apparent to
those skilled in the art upon examination of the following, or may
be learned by practice of the invention. The objects and advantages
of the invention may be realized and attained by means of the
instrumentalities and combinations particularly pointed out in the
appended claims.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0081] The accompanying drawings illustrate one or more embodiments
of the present invention and, together with the description, serve
to explain principles of the invention. The drawings are only for
the purpose of illustrating one or more preferred embodiments of
the invention and are not to be construed as limiting the
invention. In the drawings:
[0082] FIG. 1 is the sequence of wild-type endogenous human ANP
(hANP);
[0083] FIG. 2 is a graph of the concentration of construct 1-18 in
rats over time when administered by subcutaneous means at 5 mg/kg
and by intravenous means at 2 mg/kg;
[0084] FIG. 3 is a graph of total urine output over thirty minutes
in a group of four rats when administered constructs 1-18 and 1-63
by IV routes; and,
[0085] FIG. 4 is a graph of total urine output over forty-five
minutes in a group of four rats when administered construct 1-18 in
different doses by SC routes.
DETAILED DESCRIPTION OF THE INVENTION
[0086] The invention provides natriuretic receptor-specific
constructs made of a plurality of amino acid residues, at least one
ring-constrained amino acid surrogate and optionally at least one
prosthetic group. The ring-constrained amino acid surrogates
employed in the invention are preferably such that they may be made
with a conventional amino protected N-terminus, using a protecting
group such as Fmoc, and a reactive carboxyl C-terminus, and may
thus be employed in conventional peptide synthesis methodologies,
it being understood that if the amino acid surrogate is at the
C-terminus position of the construct, that other than a carboxyl
terminus may be employed on such surrogate. Thus, in a preferred
embodiment the invention provides synthetically made constructs,
synthesized using peptide synthesis methodologies modified as
appropriate, and comprising a plurality of amino acid residues and
at least one ring-constrained amino acid surrogate, In a related
preferred embodiment, the construct further includes at least one
prosthetic group.
[0087] Preferred prosthetic groups include polymeric groups
comprising repeat units including one or more carbon and hydrogen
atoms, and optionally other atoms, including oxygen. Such polymeric
groups are preferably water-soluble polymers, and are preferably
poly(alkylene oxide), poly(vinyl pyrrolidone), poly(vinyl alcohol),
polyoxazoline or poly(acryloylmorpholine). A preferred
poly(alkylene oxide) is poly(ethylene glycol) (PEG), optionally
derivatized with a linking group.
[0088] In one aspect, the invention provides a construct with an
amino acid sequence that is a homolog of a known natriuretic
peptide, such as ANP or BNP, or is a homolog of any known peptide
variant of a natriuretic peptide, wherein the construct includes at
least one amino acid surrogate of formula I or II. The
corresponding amino acid sequence not comprising an amino acid
surrogate may be identical to a known natriuretic peptide or a
known peptide variant, or may be homologous thereto, such as a
corresponding amino acid sequence that is at least 60% homologous,
or more preferably is at least about 80% homologous. As used
herein, the phrase "corresponding amino acid sequence not
comprising an amino acid surrogate" means an amino acid sequence,
including a known amino acid sequence, that binds to a receptor for
a natriuretic peptide and that does not include a surrogate. Such
known amino acid sequence is identical to the construct if the
amino acid sequence is the same but for the substitution by or
addition of one or more amino acid surrogates. Similarly, homology
is determined by reference to identity of the known amino acid
sequence to the construct but for the substitution by or addition
of one or more amino acid surrogates.
[0089] In another aspect, the invention provides a construct that
is modeled on a known peptide which binds to a receptor for a
natriuretic peptide, but which includes one or more amino acid
surrogates, such surrogates being either substituted for one or
more amino acid residues contained in the known peptide, or in
addition to the sequence comprising the known peptide. The known
peptide may be any natriuretic peptide known in the art, including
but not limited to those disclosed in any publication, patent,
application or reference cited herein, including but not limited to
the natriuretic peptides disclosed in U.S. Pat. Nos. 4,496,544;
4,609,725; 4,656,158; 4,673,732; 4,716,147; 4,757,048; 4,764,504;
4,804,650; 4,816,443; 4,824,937; 4,861,755; 4,904,763; 4,935,492;
4,952,561; 5,047,397; 5,057,495; 5,057,603; 5,091,366; 5,095,004;
5,106,834; 5,114,923; 5,159,061; 5,204,328; 5,212,286; 5,352,587;
5,376,635; 5,418,219; 5,665,704; 5,846,932; 5,583,108; 5,965,533;
6,028,055; 6,083,982; 6,124,430; 6,150,402; 6,407,211; 6,525,022;
6,586,396 or 6,818,619; in U.S. Patent Application Publications
2004/0002458; 2004/0063630; 2004/0077537; 2005/0113286;
2005/0176641; or 2006/0030004; or in various non-U.S. patents and
patent applications, including WO 85/04870; WO 85/04872; WO
88/03537; WO 88/06596; WO 89/10935; WO 89/05654; WO 90/01940; WO
90/14362; WO 92/06998; WO 95/13296; WO 99/08510; WO 99/12576; WO
01/016295; WO 2004/047871; WO 2005/072055; EPO 0 291 999; EPO 0 323
740; EPO 0 341 603; EPO 0 350 318; EPO 0 356 124; EPO 0 385 476;
EPO 0 497 368; or EPO 0 542 863. In one aspect, the known peptide
is a peptide or homolog thereof disclosed in U.S. Pat. Nos.
4,656,158, 4,824,937, 4,935,492, 5,159,061, 5,204,328, 5,376,635,
5,665,704, 5,846,932, 6,028,055, 6,407,211, 6,525,022, 6,586,396,
or 6,818,619, U.S. Patent Application Publications 2004/0002458,
2004/0063630, or 2005/0176641, or International Patent Application
Publications WO 2004/047871 or WO 2005/072055. The teachings of
each of the foregoing patents and patent applications are
incorporated by reference as if set forth in full.
[0090] In one particularly preferred embodiment, the invention
provides a construct, comprising an amino acid sequence which binds
to a natriuretic peptide receptor, wherein one or more amino acid
residues in such amino acid sequence which binds to a natriuretic
peptide receptor is substituted with an amino acid surrogate of
formula I. In one aspect, the amino acid sequence which binds to a
natriuretic peptide receptor is, prior to substitution,
H-Met-cyclo(Cys-His-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Ser-Cys)-Tyr-Arg-NH.s-
ub.2 (SEQ ID NO:1).
[0091] In yet another aspect the invention provides a construct
that binds to a receptor for a natriuretic peptide, including a
receptor for ANP or BNP, and includes at least one amino acid
surrogate of formula I or II, but which construct is not homologous
to any known peptide that binds to a receptor for a natriuretic
peptide.
[0092] In one embodiment, the invention provides a cyclic construct
of formula III:
##STR00006##
where
[0093] Aaa.sup.1 is an L- or D-isomer of an .alpha.-amino acid or
.beta.-amino acid or an .alpha., .alpha.-disubstituted amino acid
derived from an .alpha.-amino acid, including where Aaa.sup.1 is an
L- or D-isomer of an .alpha.-amino acid or .beta.-amino acid
including or derived from Nle, Ala, Leu, Ile, Val, Arg, Phe, Lys,
Tyr, Asp, Nva, Met, Met(O), or Met(O.sub.2), or an .alpha.,
.alpha.-disubstituted amino acid derived from Nle, Ala, Leu, Ile,
Val, Arg, Phe, Lys, Tyr, Asp, Nva, Met, Met(O), or Met(O.sub.2),
including all (R) or (S) configurations of .alpha.,
.alpha.-disubstituted amino acids where the substituents are
different, or Aaa.sup.1 is an acyl comprising a C.sub.2 to C.sub.18
linear alkyl, a C.sub.3 to C.sub.17 branched alkyl, a C.sub.2 to
C.sub.18 linear alkenyl or alkynyl or a C.sub.3 to C.sub.18
branched alkenyl or alkynyl, or Aaa.sup.1 is an amino acid
surrogate of the structure:
##STR00007##
wherein the broken line indicates a peptide bond; R and R' are
independently H, a linear or branched C.sub.1 to C.sub.6 aliphatic
chain, --(CH.sub.2).sub.y--S--CH.sub.3,
--(CH.sub.2).sub.y--S(.dbd.O)--CH.sub.3,
--(CH.sub.2).sub.y--S(O.sub.2)--CH.sub.3, a bond and a
cyclopropane, cyclobutane, cyclopentane, or cyclohexane ring, or a
C.sub.1 to C.sub.3 aliphatic chain and a cyclopropane, cyclobutane,
cyclopentane, or cyclohexane ring; x is 1 or 2; Y is CH.sub.2 or
C.dbd.O; W is CH.sub.2, NH or NR'''; Z is H or CH.sub.3; Q is --H,
--(CH.sub.2).sub.m--N(v.sub.3)(v.sub.4),
--(CH.sub.2).sub.m--CH.sub.3, --(CH.sub.2).sub.m--O(v.sub.3),
--(CH.sub.2).sub.m--C(.dbd.O)-(v.sub.3),
--(CH.sub.2).sub.m--C(.dbd.O)--O-(v.sub.3),
--(CH.sub.2).sub.m--S(v.sub.3),
--C(.dbd.O)--(CH.sub.2).sub.m--CH.sub.3,
--C(.dbd.O)--(CH.sub.2).sub.m--N(v.sub.3)(v.sub.4),
--C(.dbd.O)--(CH.sub.2).sub.m--C(.dbd.O)-(v.sub.3),
--C(.dbd.O)--(CH.sub.2).sub.m--O(v.sub.3), or
--C(.dbd.O)--(CH.sub.2).sub.m--S(v.sub.3); R''' is an acyl, a
C.sub.1 to C.sub.17 linear or branched alkyl chain, a C.sub.2 to
C.sub.19 linear or branched alkyl acyl chain, a C.sub.1 to C.sub.17
linear or branched omega amino aliphatic, or a C.sub.1 to C.sub.17
linear or branched omega amino aliphatic acyl; n is 0, 1 or 2; m is
0 to 17; y is 1 to 5; v.sub.3 and v.sub.4 are each independently H,
a C.sub.1 to C.sub.17 linear or branched alkyl chain or a C.sub.2
to C.sub.19 linear or branched alkyl acyl chain, on the proviso
that if one of v.sub.3 or v.sub.4 is an alkyl acyl chain, then the
other of v.sub.3 or v.sub.4 is H; and the carbon atoms marked with
an asterisk can have any stereochemical configuration;
[0094] Aaa.sup.2 and Aaa.sup.13 are the same or different, and are
each L- or D-isomer amino acid residues forming a cyclic bridge
through the side chains of each of Aaa.sup.2 and Aaa.sup.13 wherein
the linking group of the cyclic bridge is --S--S--,
--S--CH.sub.2--S--, --S--CH.sub.2--, --CH.sub.2--S--,
--C(.dbd.O)--NH--, --NH--C(.dbd.O)--, --CH.sub.2--NH--,
--NH--CH.sub.2--, --CH.sub.2--S(O).sub.n-- where n is 1 or 2,
--S(O).sub.n--CH.sub.2-- where n is 1 or 2, --CH.sub.2--CH.sub.2--,
--CH.dbd.CH-- (E or Z), --C.ident.C--, --C(.dbd.O)--O--,
--O--C(.dbd.O)--, --C(.dbd.O)--CH.sub.2--, --CH.sub.2--C(.dbd.O)--,
--O--C(.dbd.O)--NH--, --NH--C(.dbd.O)--O--, or
--NH--C(.dbd.O)--NH--;
[0095] Aaa.sup.3 is an L- or D-isomer of an .alpha.-amino acid or
.beta.-amino acid including or derived from His, Ala, Ser, Thr,
Lys, HLys, Orn, Cys, HCys, Dap, or Dab, or an .alpha.,
.alpha.-disubstituted amino acid derived from His, Ala, Ser, Thr,
Lys, HLys, Orn, Cys, HCys, Dap, or Dab, including all (R) or (S)
configurations of .alpha., .alpha.-disubstituted amino acids where
the substituents are different, or Aaa.sup.3 is an amino acid
surrogate of the structure:
##STR00008##
where R and R' are independently H or an amino acid side chain
moiety of His, Ala, Ser, Thr, Lys, HLys, Orn, Cys, HCys, Dap, or
Dab or a derivative of an amino acid side chain moiety of His, Ala,
Ser, Thr, Lys, HLys, Orn, Cys, HCys, Dap, or Dab; x is 1 or 2; Y is
CH.sub.2 or C.dbd.O; W is CH.sub.2, NH or NR'''; Z is H or
CH.sub.3; R''' is an acyl, a C.sub.1 to C.sub.17 linear or branched
alkyl chain, a C.sub.2 to C.sub.19 linear or branched alkyl acyl
chain, a C.sub.1 to C.sub.17 linear or branched omega amino
aliphatic, or a C.sub.1 to C.sub.17 linear or branched omega amino
aliphatic acyl; and n is 0, 1 or 2;
[0096] Aaa.sup.4 is an L- or D-isomer of an .alpha.-amino acid or
.beta.-amino acid including or derived from substituted or
unsubstitued Phe, HPhe or Pgl, or Tyr, Leu, Ile, Val, Ala, Nle, Nva
or Tle, or an .alpha., .alpha.-disubstituted amino acid derived
from substituted or unsubstitued Phe, HPhe or Pgl, or Tyr, Leu,
Ile, Val, Ala, Nle, Nva or Tle, including all (R) or (S)
configurations of .alpha., .alpha.-disubstituted amino acids where
the substituents are different, or Aaa.sup.4 is an amino acid
surrogate as for Aaa.sup.3 where R and R' are independently H or an
amino acid side chain moiety of substituted or unsubstitued Phe,
HPhe or Pgl, or Tyr, Leu, Ile, Val, Ala, Nle, Nva or Tle or a
derivative of an amino acid side chain moiety of substituted or
unsubstitued Phe, HPhe or Pgl, or Tyr, Leu, Ile, Val, Ala, Nle, Nva
or Tle;
[0097] Aaa.sup.5 is Gly, Sar, an L- or D-isomer of an .alpha.-amino
acid or .beta.-amino acid including or derived from Ala, or Aib,
which is the .alpha., .alpha.-disubstituted amino acid derived from
Ala, or Aaa.sup.5 is an amino acid surrogate as for Aaa.sup.3 where
R and R' are independently H or --CH.sub.3;
[0098] Aaa.sup.6 is Gly, Sar, an L- or D-isomer of an .alpha.-amino
acid or .beta.-amino acid including or derived from Ala, or Aib, or
Aaa.sup.6 is an amino acid surrogate as for Aaa.sup.3 where R and
R' are independently H or --CH.sub.3;
[0099] Aaa.sup.7 is an L- or D-isomer of an .alpha.-amino acid or
.beta.-amino acid including or derived from Arg, His, Ala, Ser,
HSer, Thr, Lys, HLys, Orn, Cys, HCys, Cit, Abu, Dap, or Dab, or an
.alpha., .alpha.-disubstituted amino acid derived from Arg, His,
Ala, Ser, HSer, Thr, Lys, HLys, Orn, Cys, HCys, Cit, Abu, Dap, or
Dab, including all (R) or (S) configurations of .alpha.,
.alpha.-disubstituted amino acids where the substituents are
different, or Aaa.sup.7 is an amino acid surrogate as for Aaa.sup.3
where R and R' are independently H or an amino acid side chain
moiety of Arg, His, Ala, Ser, HSer, Thr, Lys, HLys, Orn, Cys, HCys,
Abu, Dap, or Dab or a derivative of an amino acid side chain moiety
of Arg, His, Ala, Ser, HSer, Thr, Lys, HLys, Orn, Cys, HCys, Abu,
Dap, or Dab;
[0100] Aaa.sup.8 is Gly, an L- or D-isomer of an .alpha.-amino acid
or .beta.-amino acid including or derived from Nle, Ile, Leu, Val,
Phe, Ala, Nva, Met(O), Met(O.sub.2), or Tle, or an .alpha.,
.alpha.-disubstituted amino acid derived from Nle, Ile, Leu, Val,
Phe, Ala, Nva, Met(O), Met(O.sub.2), or Tle, including all (R) or
(S) configurations of .alpha., .alpha.-disubstituted amino acids
where the substituents are different, or Aaa.sup.8 is an amino acid
surrogate as for Aaa.sup.3 where R and R' are independently H or an
amino acid side chain moiety of Nle, Ile, Leu, Val, Phe, Ala, Nva,
Met(O), Met(O.sub.2), or Tle, or a derivative of an amino acid side
chain moiety of Nle, Ile, Leu, Val, Phe, Ala, Nva, Met(O),
Met(O.sub.2), or Tle;
[0101] Aaa.sup.9 is an L- or D-isomer of an .alpha.-amino acid or
.beta.-amino acid including or derived from Asp, Glu, His, Ala,
Ser, Thr, Lys, HLys, Cys, HCys, Met(O), Met(O.sub.2), Orn, Dap, or
Dab, or an .alpha., .alpha.-disubstituted amino acid derived from
Asp, Glu, His, Ala, Ser, Thr, Lys, HLys, Cys, HCys, Met(O),
Met(O.sub.2), Orn, Dap, or Dab, including all (R) or (S)
configurations of .alpha., .alpha.-disubstituted amino acids where
the substituents are different, or Aaa.sup.9 is an amino acid
surrogate as for Aaa.sup.3 where R and R' are independently H or an
amino acid side chain moiety of Asp, Glu, His, Ala, Ser, Thr, Lys,
HLys, Cys, HCys, Met(O), Met(O.sub.2), Orn, Dap, or Dab or a
derivative of an amino acid side chain moiety of Asp, Glu, His,
Ala, Ser, Thr, Lys, HLys, Cys, HCys, Met(O), Met(O.sub.2), Orn,
Dap, or Dab;
[0102] Aaa.sup.10 is an L- or D-isomer of an .alpha.-amino acid or
.beta.-amino acid including or derived from Arg, His, Ala, Ser,
Thr, Lys, HLys, Cys, HCys, Cit, Met(O), Orn, Dap, or Dab, or an
.alpha., .alpha.-disubstituted amino acid derived from Arg, His,
Ala, Ser, Thr, Lys, HLys, Cys, HCys, Cit, Met(O), Orn, Dap, or Dab,
including all (R) or (S) configurations of .alpha.,
.alpha.-disubstituted amino acids where the substituents are
different, or Aaa.sup.10 is an amino acid surrogate as for
Aaa.sup.3 where R and R' are independently H or an amino acid side
chain moiety of Arg, His, Ala, Ser, Thr, Lys, HLys, Cys, HCys,
Met(O), Orn, Dap, or Dab or a derivative of an amino acid side
chain moiety of Arg, His, Ala, Ser, Thr, Lys, HLys, Cys, HCys,
Met(O), Orn, Dap, or Dab;
[0103] Aaa.sup.11 is Gly or a D- or L-isomer of an .alpha.-amino
acid or .beta.-amino acid including or derived from Nle, Ile, Leu,
Val, Phe, Ala, Nva, Cys, HCys, Abu or Tle, or an .alpha.,
.alpha.-disubstituted amino acid derived from Nle, Ile, Leu, Val,
Phe, Ala, Nva, Cys, HCys, Abu or Tle, including all (R) or (S)
configurations of .alpha., .alpha.-disubstituted amino acids where
the substituents are different, or Aaa.sup.11 is an amino acid
surrogate as for Aaa.sup.3 where R and R' are independently H or an
amino acid side chain moiety of Nle, Ile, Leu, Val, Phe, Ala, Nva,
Cys, HCys, Abu or Tle or a derivative of an amino acid side chain
moiety of Nle, Ile, Leu, Val, Phe, Ala, Nva, Cys, HCys, Abu or
Tle;
[0104] Aaa.sup.12 is Gly, an L- or D-isomer of an .alpha.-amino
acid or .beta.-amino acid including or derived from Ser, Nle, Ile,
Leu, Val, Phe, Ala, Nva, Arg, Lys, Orn, Cys, HCys, Abu or Tle, or
an .alpha., .alpha.-disubstituted amino acid derived from Ser, Nle,
Ile, Leu, Val, Phe, Ala, Nva, Arg, Lys, Orn, Cys, HCys, Abu or Tle,
including all (R) or (S) configurations of .alpha.,
.alpha.-disubstituted amino acids where the substituents are
different, or Aaa.sup.12 is an amino acid surrogate as for
Aaa.sup.3 where R and R' are independently H or an amino acid side
chain moiety of Ser, Nle, Ile, Leu, Val, Phe, Ala, Nva, Arg, Lys,
Orn, Cys, HCys, Abu or Tle or a derivative of an amino acid side
chain moiety of Ser, Nle, Ile, Leu, Val, Phe, Ala, Nva, Arg, Lys,
Orn, Cys, HCys, Abu or Tle;
[0105] Aaa.sup.14 is an L- or D-isomer of an .alpha.-amino acid or
.beta.-amino acid including or derived from substituted or
unsubstitued Phe, HPhe or Pgl, or Tyr, Leu, Ile, Val, Ala, Lys,
Orn, Nle, Nva or Tle, or an .alpha., .alpha.-disubstituted amino
acid derived from substituted or unsubstitued Phe, HPhe or Pgl, or
Tyr, Leu, Ile, Val, Ala, Lys, Orn, Nle, Nva or Tle, including all
(R) or (S) configurations of .alpha., .alpha.-disubstituted amino
acids where the substituents are different, or Aaa.sup.14 is an
amino acid surrogate of the structure of formula II as for
Aaa.sup.3 where R and R' are independently H or an amino acid side
chain moiety of substituted or unsubstitued Phe, HPhe or Pgl, or
Tyr, Leu, Ile, Val, Ala, Lys, Orn, Nle, Nva or Tle or a derivative
of an amino acid side chain moiety of substituted or unsubstitued
Phe, HPhe or Pgl, or Tyr, Leu, Ile, Val, Ala, Lys, Orn, Nle, Nva or
Tle; and
[0106] Aaa.sup.15 is a D- or L-isomer of an .alpha.-amino acid or
.beta.-amino acid including or derived from Ala, Arg, Orn, Lys,
Ala, Dap, Dab, HArg, or HLys, or an .alpha., .alpha.-disubstituted
amino acid derived from Ala, Arg, Orn, Lys, Ala, Dap, Dab, HArg, or
HLys, including all (R) or (S) configurations of .alpha.,
.alpha.-disubstituted amino acids where the substituents are
different, or Aaa.sup.15 is an amino acid surrogate of the
structure:
##STR00009##
wherein the broken line indicates a peptide bond; at least one of R
and R' is --(CH.sub.2).sub.y--R'' and if one, the remaining of R
and R' is H, where R'' is: [0107] --NH.sub.2, [0108]
--NH--C(.dbd.NH)--NH.sub.2, [0109]
--NH--(CH.sub.2).sub.y--NH.sub.2, [0110] --NH--C(.dbd.O)--NH.sub.2,
[0111] --C(.dbd.O)--NH.sub.2, [0112] --C(.dbd.O)--NH--CH.sub.3,
[0113] --C(.dbd.O)--NH--(CH.sub.2).sub.y--NH.sub.2, [0114]
--NH--C(.dbd.NH)--NH-Me, [0115] --NH--C(.dbd.NH)--NH-Et, [0116]
--NH--C(.dbd.NH)--NH--Pr, [0117] --NH--C(.dbd.NH)--NH--Pr-i, [0118]
--NH--C(.dbd.O)--CH.sub.3, [0119]
--NH--C(.dbd.O)--CH.sub.2--CH.sub.3, [0120]
--NH--C(.dbd.O)--CH--(CH.sub.3).sub.2, [0121]
--NH--C(.dbd.O)--O--CH.sub.3, [0122]
--NH--C(.dbd.O)--O--CH.sub.2--CH.sub.3, [0123]
--NH--C(.dbd.O)--O--C--(CH.sub.3).sub.3, [0124]
--NH--C(.dbd.O)--NH--CH.sub.3, [0125]
--NH--C(.dbd.N--C(.dbd.O)--O--C--(CH.sub.3).sub.3)--NH--C(.dbd.O)--O--C---
(CH.sub.3).sub.3, [0126]
--N(C(.dbd.O)--O--C--(CH.sub.3).sub.3)--C(.dbd.NH)--NH--C(.dbd.O)--O--C---
(CH.sub.3).sub.3,
##STR00010##
[0126] x is 1 or 2; Y is CH.sub.2 or C.dbd.O; W is CH.sub.2, NH or
NR'''; Z is H or CH.sub.3; J is --H, --(CH.sub.2).sub.m--OH,
--C(.dbd.O)--CH.sub.2).sub.m--OH,
--C(.dbd.O)--CH.sub.2).sub.m--N(v.sub.1)(v.sub.2),
--C(.dbd.O)--O--(CH.sub.2).sub.m--CH.sub.3,
--O--(CH.sub.2).sub.m--CH.sub.3,
--O--(CH.sub.2).sub.m--N(v.sub.1)(v.sub.2),
--O--(CH.sub.2).sub.m--OH,
--C(.dbd.O)--NH--(CH.sub.2).sub.m--CH.sub.3,
--C(.dbd.O)--NH--(CH.sub.2).sub.m--N(v.sub.1)(v.sub.2),
--C(.dbd.O)--NH--(CH.sub.2).sub.m--S(v.sub.1),
--C(.dbd.O)--N--((CH.sub.2).sub.m--N(v.sub.1)(v.sub.2)).sub.2,
--C(.dbd.O)--NH--CH(--C(.dbd.O)--OH)--(CH.sub.2).sub.m--N(v.sub.1)(v.sub.-
2),
--C(.dbd.O)--NH--(CH.sub.2).sub.m--NH--C(.dbd.O)--CH(N(v.sub.1)(v.sub.-
2))((CH.sub.2).sub.m--N(v.sub.1)(v.sub.2)),
--C(.dbd.O)--NH--CH(--C(.dbd.O)--N(v.sub.1)(v.sub.2))--(CH.sub.2).sub.m---
N(v.sub.1)(v.sub.2), an omega amino aliphatic, terminal aryl or
aralkyl group, any single natural or unnatural .alpha.-amino acid,
.beta.-amino acid or .alpha., .alpha.-disubstituted amino acid in
combination with one of the foregoing groups defining J, or any
single natural or unnatural .alpha.-amino acid, .beta.-amino acid
or .alpha., .alpha.-disubstituted amino acid, including all (R) and
(S) configurations of any of the foregoing; R''' is an acyl, a
C.sub.1 to C.sub.17 linear or branched alkyl chain, a C.sub.2 to
C.sub.19 linear or branched alkyl acyl chain, a C.sub.1 to C.sub.17
linear or branched omega amino aliphatic, or a C.sub.1 to C.sub.17
linear or branched omega amino aliphatic acyl; v.sub.1 and v.sub.2
are each independently H or a C.sub.1 to C.sub.17 linear or
branched alkyl chain; n is 0, 1 or 2; m is 0 to 17; y is 1 to 5;
and the carbon atoms marked with an asterisk can have any
stereochemical configuration;
[0127] on the proviso that at least one of Aaa.sup.1, Aaa.sup.3
through Aaa.sup.12, Aaa.sup.14 or Aaa.sup.15 is an amino acid
surrogate.
[0128] A related embodiment of formula III provides a construct
where one or more of Aaa.sup.1, Aaa.sup.3 to Aaa.sup.12, Aaa.sup.14
or Aaa.sup.15 is an amino acid surrogate as defined above, and
where a prosthetic group, as hereafter defined, is attached to a
reactive group of a side chain of an amino acid residue at one or
more of Aaa.sup.1, Aaa.sup.3 to Aaa.sup.12, Aaa.sup.14 or
Aaa.sup.15, to a reactive R or R' group of an amino acid surrogate
at Aaa.sup.3 to Aaa.sup.12 or Aaa.sup.14, directly or through a Q
group to the terminal amine of an amino acid surrogate at
Aaa.sup.1, to a reactive terminal carboxyl of an amino acid
surrogate at Aaa.sup.5, or to a reactive group forming a part of J
of an amino acid surrogate at Aaa.sup.15. The reactive group to
which the one or more prosthetic groups are covalently bonded may
be a primary amine, a secondary amine, a carboxyl group, a thiol
group or a hydroxyl group. In one aspect, the prosthetic group may
be covalently bound to a reactive amine in position Aaa.sup.1,
Aaa.sup.3, Aaa.sup.7, Aaa.sup.10, Aaa.sup.12 or Aaa.sup.15 or a
combination of the foregoing. In another aspect, the prosthetic
group may be covalently bound to a reactive carboxyl in position
Aaa.sup.9 or Aaa.sup.15 or both. In another aspect, the prosthetic
group may be covalently bound to a reactive thiol in position
Aaa.sup.3, Aaa.sup.7, Aaa.sup.9, Aaa.sup.10, Aaa.sup.11, or
Aaa.sup.12 or a combination of the foregoing.
[0129] In a preferred aspect of the construct of formula III, one,
two or three of Aaa.sup.1 to Aaa.sup.15 (excluding Aaa.sup.2 and
Aaa.sup.13) are an amino acid surrogate of one of the foregoing
formulas. In a first particularly preferred aspect, one of
Aaa.sup.1, Aaa.sup.5 and Aaa.sup.15 is an amino acid surrogate. In
a second particularly preferred aspect, two of Aaa.sup.1, Aaa.sup.5
and Aaa.sup.15 are amino acid surrogates. In a third particularly
preferred aspect, each of Aaa.sup.1, Aaa.sup.5 and Aaa.sup.15 are
amino acid surrogates. In another particularly preferred aspect,
one, two or three of Aaa.sup.1, Aaa.sup.5 and Aaa.sup.15 are amino
acid surrogates, and the construct is a cyclic construct formed by
disulfide bond formation through the side chains of Aaa.sup.2 and
Aaa.sup.13. In another particularly preferred aspect, where two or
more of Aaa.sup.1 to Aaa.sup.15 are amino acid surrogates the amino
acid surrogates are not contiguous, which is to say that each amino
acid surrogate is separate from each other amino acid surrogate by
at least one amino acid residue being interposed therebetween in
the primary sequence.
[0130] In yet another preferred embodiment, in the construct of
formula III at least one of Aaa.sup.3, Aaa.sup.5, Aaa.sup.6,
Aaa.sup.7, Aaa.sup.9, Aaa.sup.10, or Aaa.sup.12 is an L- or
D-isomer of Ala, preferably an L-isomer of Ala.
[0131] In yet another embodiment, the invention provides a
construct of formula III further comprising one or more non-peptide
bonds. Non-peptide bonds may be employed to decrease the
susceptibility of a construct of the invention to degradation, such
as improving the in vivo stability of constructs towards
tryptic-like proteases by replacing the native peptide bond before
each Lys or Arg residue with a non-peptide bond, such as an
isostere of an amide, a substituted amide or a peptidomimetic
linkage. In one specific embodiment, native peptide bonds are
replaced with peptide bonds having a reversed polarity. In general,
any non-peptide bond may be employed, and may be utilized between
any two residues. A non-peptide bond includes bonds in which the
carbon atom participating in the bond between two residues is
reduced from a carbonyl carbon to a methylene carbon, such as a
non-peptide bond --CH.sub.2--NH-- or its isostere --NH--CH.sub.2--,
or the use of other bonds such as --CH.sub.2--S--, --CH.sub.2--O--,
or --C(.dbd.O)--CH.sub.2-- or an isostere of any of the foregoing,
or --CH.sub.2--CH.sub.2-- or --CH.dbd.CH--. In general, non-peptide
bonds include an imino, ester, hydrazine, semicarbazide, oxime, or
azo bond.
[0132] The constructs defined above may include one or more
prosthetic groups. Prosthetic groups may be employed to modulate
the residence time in circulation, to modulate bioavailability,
modulate immunogenicity of constructs, or the like. In general,
prosthetic groups "modulate" by increasing the residence time,
bioavailability or the like, as the case may be, but prosthetic
groups may optionally decrease residence time, bioavailability or
the like. A "prosthetic group" thus includes any compound
conjugated, such as by a covalent bond, to a construct of any
formula, for purposes of improving pharmacokinetic or
pharmacodynamic properties of the construct. Preferred prosthetic
groups include polymeric groups, comprising repeat units which in
turn comprise one or more carbon and hydrogen atoms, and optionally
other atoms, including oxygen atoms. Such polymeric groups are
preferably water-soluble polymers, and are preferably poly(alkylene
oxide), poly(vinyl pyrrolidone), poly(vinyl alcohol), polyoxazoline
or poly(acryloylmorpholine). A preferred poly(alkylene oxide) is
poly(ethylene glycol) (PEG). In addition to PEG, other
poly(alkylene glycol) polymers may be employed, such as
poly(propylene glycol) and poly(butylene glycol).
[0133] In one embodiment, the prosthetic group is one or more PEG
polymers covalently bound to a reactive group of the construct. The
PEG polymer, or other prosthetic group, may be covalently bound to
a reactive group on the side chain of one or more amino acid
residues, or may be covalently bound to a reactive group on an
amino acid surrogate. Such reactive groups of an amino acid
surrogate may include a group covalently bound, directly or through
one or more intermediates, to Q or J, or may include a reactive
group forming a part of R or R'.
[0134] If PEG is employed as the prosthetic group, the PEG polymer
may have a molecular weight of from about 200 MW to about 50000 MW.
The PEG polymer may be linear, and if linear, may be
monofunctional, with a reactive group at one end and a non-reactive
group at the other end, homobifunctional, with the same reactive
group at each end, or heterobifunctional, with a different reactive
group at each end. Alternatively, the PEG polymer may be branched,
having generally a "Y"-shaped configuration, multi-armed, such as
with two, three, four or eight arms, or other configurations known
in the art. The PEG polymer preferably has at least one derivatized
reactive group for linking to one or more defined groups on the
construct of any of formula III through XIII, preferably by means
of a covalent bond. The derivativized reactive group may link to,
for example, an amine, hydroxyl, thiol, or carboxyl group on a
construct, including on a terminal group of an amino acid residue,
on a side chain of an amino acid residue, on a Q group of a
surrogate, on a J group of a surrogate, or on an R or R' group of a
surrogate.
[0135] The PEG polymer preferably has, at one end, an end-cap
group, such as a hydroxyl, alkoxy, substituted alkoxy, aleknoxy,
substituted alkenoxy, alkynoxy, substituted alkynoxy, aryloxy or
substituted aryloxy. The PEG polymer further preferably has, at
least one other end, a derivatized reactive group. In one
embodiment, the PEG polymer is a linear or branched polyether with
a terminal hydroxyl group, such as a monomethoxy PEG, which is
derivatized with a linking group, such as an amine, maleimide or
carboxylic acid. The available reactive groups of the construct
dictate the derivatized linking group employed on the PEG polymer.
Thus, in one embodiment, the N-terminal amine of the construct is
employed, using a carboxylic acid derivatized PEG. In another
embodiment, the C-terminal amine of the construct is employed,
again using a carboxylic acid derivatized PEG. In yet another
embodiment, if a Lys residue or homolog thereof is present in the
construct, either the .alpha. or .epsilon. amino group thereof may
be employed, again using a carboxylic acid derivatized PEG.
Maleimide derivatized PEG may be employed with either a reactive
thiol or hydroxyl group on the construct. Similarly, amine
derivatized PEG may be employed with a reactive carboxyl group on
any terminal group or side chain of an amino acid residue, on a Q
group of a surrogate, on a J group of a surrogate, or on an R or R'
group of a surrogate.
[0136] Thus, in one aspect, PEG is activiated with one or more
electrophilic groups and may be employed for coupling to amino
groups of the construct, including coupling to an E amino group of
a side chain or an N-terminal or C-terminal amine. Representative
electrophilic reactive groups include succinimidyl
.alpha.-methylbutanoate and other .alpha.-methylbutyric acid
esters, as disclosed in U.S. Pat. Nos. 5,672,662 and 6,737,505, and
may be be used with proteins, as disclosed in U.S. Patent
Application Publication 2004/0235734. Alternatively, succinimidyl
propionate may be employed as a reactive group, as disclosed in
U.S. Pat. No. 5,567,662, or N-hydroxysuccinimide may be employed
with a branched PEG, as disclosed in U.S. Pat. No. 5,932,462. The
teachings of each of the foregoing patents and patent applications
are incorporated by reference as if set forth in full.
[0137] In another aspect, PEG polymers are provided with one or
more reactive aldehyde groups, and employed for coupling to a
terminal primary amine, such as an N-terminal or C-terminal amine.
In another aspect, PEG polymers are provided with one or more
thiol-reactive groups, such as a maleimide, ortho-pyridyldisulfide,
or thiol group, and are employed for coupling to a reactive thiol
in the construct of any of formula III through XIII, such as a
reactive thiol in a cysteine side chain or a reactive thiol in a Q
group of a construct.
[0138] In one aspect, any of the methods, conjugates or schemes as
disclosed in International Patent Publication No. WO 2004/047871,
or any reference cited therein, may be employed with the constructs
of this invention. The teaching of the foregoing patent
applications is incorporated by reference as if set forth in
full.
[0139] In general, some form of chemical modification may be
employed to make an active PEG derivative with a reactive group.
The reactive group may be an active carbonate, an active ester, an
aldehyde, or tresylate. In part, the reactive group of the PEG
determines the amino acid terminal group or side chain moiety to
which the PEG derivative is bound. In general, site specific
PEGylation is preferred, in part because the resulting construct is
homogeneous, minimizing loss of biological activity and reducing
immunogenicity.
[0140] In one embodiment, the PEG has a molecular weight of from
about 200 MW to about 50,000 MW, more preferably from about 2,000
MW to about 20,000 MW. In another embodiment, monomethoxy PEG, such
as of the formula
CH.sub.3--O(CH.sub.2--CH.sub.2--O).sub.n--CH.sub.2--CH.sub.2--OH or
CH.sub.3--O(CH.sub.2--CH.sub.2--O).sub.n--H, where n is any integer
from 2 to about 1200, is employed, preferably derivatized with an
amine, maleimide or carboxylic acid linking group.
[0141] In another embodiment, the prosthetic group, such as PEG, is
conjugated to a construct by means of an enzymatically labile
linker as described in Veronese F M and Pasut G. Pegylation,
successful approach to drug delivery. Drug Discovery Today
10:1451-1458 (2005), and the methods disclosed therein are
incorporated here by reference.
[0142] In another embodiment, the prosthetic group employed is a
polymer with both a lipophilic moiety and a hydrophilic polymer
moiety, as disclosed in U.S. Pat. Nos. 5,359,030 and 5,681,811. In
a related embodiment, the prosthetic group employed is an oligomer
conjugate with a hydrophilic component, such as a PEG polymer, and
a lipophilic component, such as a branched fatty acid or alkyl
chain, linked by a hydrolyzable bond, such as an ester bond, as
disclosed in U.S. Pat. No. 6,309,633. In another related
embodiment, the prosthetic group employed is an oligomer that
includes poly(propylene glycol), and preferably at least two
poly(propylene glycol) subunits, as disclosed in U.S. Pat. No.
6,858,580. The teachings of each of the foregoing patents and
patent applications are incorporated by reference as if set forth
in full.
[0143] In yet another embodiment, the teachings of U.S. Published
Patent Application 2004/0203081 are incorporated here by reference,
including specifically teachings relating to prosthetic groups,
referred to in such application as "modifying moieties," attached
to various natriuretic compounds, and specifically oligomeric
structures having a variety of lengths and configurations. In a
related embodiment, the teachings of International Patent
Publication WO 2004/047871 are incorporated by reference, including
teachings related to "modifying moieties" attached by means of
"modifying moiety conjugation sites" to natriuretic molecules
binding to NPRA, it being understood that similar methods could be
employed with natriuretic molecules binding to other natriuretic
receptors.
[0144] Certain terms as used throughout the specification and
claims are defined as follows.
[0145] The "construct" and "amino acid residue sequences" of this
invention can be a) naturally-occurring, b) produced by chemical
synthesis, c) produced by recombinant DNA technology, d) produced
by biochemical or enzymatic fragmentation of larger molecules, e)
produced by methods resulting from a combination of methods a
through d listed above, or f) produced by any other means for
producing peptides or amino acid sequences.
[0146] By employing chemical synthesis, a preferred means of
production, it is possible to introduce various amino acids which
do not naturally occur into the construct, modify the N- or
C-terminus, and the like, thereby providing for improved stability
and formulation, resistance to protease degradation, and the like,
and to introduce one or more amino acid surrogates into the
construct.
[0147] The term "peptide" as used throughout the specification and
claims is intended to include any structure comprised of two or
more amino acids, including chemical modifications and derivatives
of amino acids. The amino acids forming all or a part of a peptide
may be naturally occurring amino acids, stereoisomers and
modifications of such amino acids, non-protein amino acids,
post-translationally modified amino acids, enzymatically modified
amino acids, and the like. The term "peptide" also includes dimers
or multimers of peptides. A "manufactured" peptide includes a
peptide produced by chemical synthesis, recombinant DNA technology,
biochemical or enzymatic fragmentation of larger molecules,
combinations of the foregoing or, in general, made by any other
method.
[0148] The term "amino acid side chain moiety" used in this
invention, including as used in the specification and claims,
includes any side chain of any amino acid, as the term "amino acid"
is defined herein. This thus includes the side chain moiety present
in naturally occurring amino acids. It further includes side chain
moieties in modified naturally occurring amino acids, such as
glycosylated amino acids. It further includes side chain moieties
in stereoisomers and modifications of naturally occurring protein
amino acids, non-protein amino acids, post-translationally modified
amino acids, enzymatically synthesized amino acids, derivatized
amino acids, constructs or structures designed to mimic amino
acids, and the like. For example, the side chain moiety of any
amino acid disclosed herein is included within the definition. A
"derivative of an amino acid side chain moiety" as hereafter
defined is included within the definition of an amino acid side
chain moiety.
[0149] The "derivative of an amino acid side chain moiety" is a
modification to or variation in any amino acid side chain moiety,
including a modification to or variation in either a naturally
occurring or unnatural amino acid side chain moiety, wherein the
modification or variation includes: (a) adding one or more
saturated or unsaturated carbon atoms to an existing alkyl, aryl,
or aralkyl chain; (b) substituting a carbon in the side chain with
another atom, preferably oxygen or nitrogen; (c) adding a terminal
group to a carbon atom of the side chain, including methyl
(--CH.sub.3), methoxy (--OCH.sub.3), nitro (--NO.sub.2), hydroxyl
(--OH), or cyano (--C.ident.N); (d) for side chain moieties
including a hydroxy, thiol or amino groups, adding a suitable
hydroxy, thiol or amino protecting group; or (e) for side chain
moieties including a ring structure, adding one or ring
substituents, including hydroxyl, halogen, alkyl, or aryl groups
attached directly or through an ether linkage. For amino groups,
suitable amino protecting groups include, but are not limited to,
Z, Fmoc, Boc, Pbf, Pmc and the like.
[0150] The "amino acids" used in embodiments of the present
invention, and the term as used in the specification and claims,
include the known naturally occurring protein amino acids, which
are referred to by both their common three letter abbreviation and
single letter abbreviation. See generally Synthetic Peptides: A
User's Guide, G. A. Grant, editor, W.H. Freeman & Co., New York
(1992), the teachings of which are incorporated herein by
reference, including the text and table set forth at pages 11
through 24. An "amino acid" includes conventional .alpha.-amino
acids and further includes .beta.-amino acids, .alpha.,
.alpha.-disubstituted amino acids and N-substituted amino acids
wherein at least one side chain is an amino acid side chain moiety
as defined herein. An "amino acid" further includes N-alkyl
.alpha.-amino acids, wherein the N-terminus amino group has a
C.sub.1 to C.sub.6 linear or branched alkyl substituent. It may
thus be seen that the term "amino acid" includes stereoisomers and
modifications of naturally occurring protein amino acids,
non-protein amino acids, post-translationally modified amino acids,
enzymatically synthesized amino acids, derivatized amino acids,
constructs or structures designed to mimic amino acids, and the
like. Modified and unusual amino acids are described generally in
Synthetic Peptides: A User's Guide, cited above; Hruby V. J.,
Al-obeidi F., Kazmierski W., Biochem. J. 268:249-262 (1990); and
Toniolo C., Int. J. Peptide Protein Res. 35:287-300 (1990); the
teachings of all of which are incorporated herein by reference. In
addition, the following abbreviations, including amino acids and
protecting and modifying groups thereof, have the meanings given:
[0151] Abu--gamma-amino butyric acid [0152] 12-Ado--12-amino
dodecanoic acid [0153] Aib--alpha-aminoisobutyric acid [0154]
6-Ahx--6-amino hexanoic acid [0155]
Amc--4-(aminomethyl)-cyclohexane carboxylic acid [0156]
8-Aoc--8-amino octanoic acid [0157] Bip--biphenylalanine [0158]
Boc--t-butoxycarbonyl [0159] Bzl--benzyl [0160] Bz--benzoyl [0161]
Cit--citrulline [0162] Dab--diaminobutyric acid [0163]
Dap--diaminopropionic acid [0164] Dip--3,3-diphenylalanine [0165]
Disc--1,3-dihydro-2-H-isoindolecarboxylic acid [0166] Et--ethyl
[0167] Fmoc--fluorenylmethoxycarbonyl [0168]
Hept--heptanoyl(CH.sub.3--(CH.sub.2).sub.5--C(.dbd.O)--) [0169]
Hex--hexanoyl(CH.sub.3--(CH.sub.2).sub.4--C(.dbd.O)--) [0170]
HArg--homoarginine [0171] HCys--homocysteine [0172]
HLys--homolysine [0173] HPhe--homophenylalanine [0174]
HSer--homoserine [0175] Me--methyl [0176] Met(O)--methionine
sulfoxide [0177] Met(O.sub.2)--methionine sulfone [0178]
Nva--norvaline [0179] Pgl--phenylglycine [0180] Pr--propyl [0181]
Pr-isopropyl [0182] Sar--sarcosine [0183] Tle--tert-butylalanine
[0184] z--benzyloxycarbonyl
[0185] In the listing of constructs according to the present
invention, conventional amino acid residues have their conventional
meaning as given in Chapter 2400 of the Manual of Patent Examining
Procedure, 8.sup.th Ed. Thus, "Nle" is norleucine; "Asp" is
aspartic acid; "His" is histidine; "Arg" is arginine; "Trp" is
tryptophan; "Lys" is lysine; "Gly" is glycine; "Pro" is proline;
"Tyr" is tyrosine, "Ser" is serine and so on. All residues are in
the L-isomer configuration unless the D-isomer is specified, as in
"D-Ala" for D-alanine.
[0186] A single amino acid, including stereoisomers and
modifications of naturally occurring protein amino acids,
non-protein amino acids, post-translationally modified amino acids,
enzymatically synthesized amino acids, derivatized amino acids, an
.alpha., .alpha.-disubstituted amino acid derived from any of the
foregoing (i.e., an .alpha., .alpha.-disubstituted amino acid
wherein at least one side chain is the same as that of the residue
from which it is derived), a .beta.-amino acid derived from any of
the foregoing (i.e., a .beta.-amino acid which other than for the
presence of a .beta.-carbon is otherwise the same as the residue
from which it is derived) and the like, including all of the
foregoing, is sometimes referred to herein as a "residue."
[0187] An ".alpha., .alpha.-disubstituted amino acid" includes any
.alpha.-amino acid having a further substituent in the
.alpha.-position, which substituent may be the same as or different
from the side chain moiety of the .alpha.-amino acid. Suitable
substituents, in addition to the side chain moiety of the
.alpha.-amino acid, include C.sub.1 to C.sub.6 linear or branched
alkyl. Aib is an example of an .alpha., .alpha.-disubstituted amino
acid. While .alpha., .alpha.-disubstituted amino acids can be
referred to using conventional L- and D-isomeric references, it is
to be understood that such references are for convenience, and that
where the substituents at the .alpha.-position are different, such
amino acid can interchangeably be referred to as an .alpha.,
.alpha.-disubstituted amino acid derived from the L- or D-isomer,
as appropriate, of a residue with the designated amino acid side
chain moiety. Thus (S)-2-Amino-2-methyl-hexanoic acid can be
referred to as either an .alpha., .alpha.-disubstituted amino acid
derived from L-Nle or as an .alpha., .alpha.-disubstituted amino
acid derived from D-Ala. Whenever an .alpha., .alpha.-disubstituted
amino acid is provided, it is to be understood as including all (R)
and (S) configurations thereof.
[0188] An "N-substituted amino acid" includes any amino acid
wherein an amino acid side chain moiety is covalently bonded to the
backbone amino group, optionally where there are no substituents
other than H in the .alpha.-carbon position. Sarcosine is an
example of an N-substituted amino acid. By way of example,
sarcosine can be referred to as an N-substituted amino acid
derivative of Ala, in that the amino acid side chain moiety of
sarcosine and Ala is the same, methyl.
[0189] The term "amino acid surrogate" includes a molecule
disclosed herein which is a mimic of a residue, including but not
limited to piperazine core molecules, keto-piperazine core
molecules and diazepine core molecules. Unless otherwise specified,
an amino acid surrogate is understood to include both a carboxyl
group and amino group, and a group corresponding to an amino acid
side chain, or in the case of an amino acid surrogate of glycine,
no side chain other than hydrogen. Thus an amino acid surrogate
includes a molecule of the general formula of formula I or II given
above. An amino acid surrogate further includes molecules of any of
the following structures, it being understood that for convenience
such structures are given as the isolated surrogate, not including
any protecting group and not bound by one or two peptide bonds to
one or two amino acid residues forming a part of a construct of the
invention:
##STR00011##
where R, R', x and the asterisks are as defined for the surrogate
of formula I. An amino acid surrogate further includes molecules of
any of the following structures, again it being understood that for
convenience such structures are given as the isolated surrogate,
not including any protecting group and not bound by one or two
peptide bonds to one or two amino acid residues forming a part of a
construct of the invention:
##STR00012##
where R, R', x and the asterisks are as defined for the surrogate
of formula I. For purposes of synthesis, either the carboxyl group
or the amino group of any amino acid surrogate is preferably
protected by a protecting group, such that it is not reactive while
the protecting group is present, and similarly any reactive group
forming a part of R or R' may similarly be protected by a
protecting group. It will be appreciated that the surrogates of the
present invention have more than one asymmetric center, and
therefore are capable of existing in more than one stereoisomeric
form. Some of the compounds may also exist as geometric isomers and
rotamers. Furthermore, some compounds of the invention may also
have conformational axial chirality resulting in atropisomers. The
invention extends to each of these forms individually and to
mixtures thereof, including racemates. In one aspect, surrogate
isomers may be separated conventionally by chromatographic methods
or by use of a resolving agent. In another aspect, individual
surrogate isomers, or enantiomerically pure surrogates, are
prepared by synthetic schemes, such as those disclosed herein or
variants of such schemes, employing asymmetric synthesis using
chiral intermediates, reagents or catalysts.
[0190] The term "C-terminus capping group" includes any terminal
group attached through the terminal ring carbon atom or, if
provided, terminal carboxyl group, of the C-terminus of a
construct. The terminal ring carbon atom or, if provided, terminal
carboxyl group, may form a part of a residue, or may form a part of
an amino acid surrogate. In a preferred aspect, the C-terminus
capping group forms a part of an amino acid surrogate which is at
the C-terminus position of the construct. The C-terminus capping
group includes, but is not limited to, --(CH.sub.2).sub.n--OH,
--(CH.sub.2), --C(.dbd.O)--OH, --(CH.sub.2).sub.m--OH,
--(CH.sub.2), --C(.dbd.O)--N(v.sub.1)(v.sub.2),
--(CH.sub.2).sub.n--C(.dbd.O)--(CH.sub.2).sub.m--N(v.sub.1)(v.sub.2),
--(CH.sub.2).sub.n--O--(CH.sub.2).sub.m--CH.sub.3,
--(CH.sub.2).sub.n--C(.dbd.O)--NH--(CH.sub.2).sub.m--CH.sub.3,
--(CH.sub.2).sub.n--C(.dbd.O)--NH--(CH.sub.2).sub.m--N(v.sub.1)(v.sub.2),
--(CH.sub.2).sub.n--C(.dbd.O)--N--((CH.sub.2).sub.m--N(v.sub.1)(v.sub.2))-
.sub.2,
--(CH.sub.2).sub.n--C(.dbd.O)--NH--CH(--C(.dbd.O)--OH)--(CH.sub.2)-
.sub.m--N(v.sub.1)(v.sub.2),
--C(.dbd.O)--NH--(CH.sub.2).sub.m--NH--C(.dbd.O)--CH(N(v.sub.1)(v.sub.2))-
((CH.sub.2).sub.m--N(v.sub.1)(v.sub.2)), or
--(CH.sub.2).sub.n--C(.dbd.O)--NH--CH(--C(.dbd.O)--NH.sub.2)--(CH.sub.2).-
sub.m--N(v.sub.1)(v.sub.2), including all (R) or (S) configurations
of the foregoing, where v.sub.1 and v.sub.2 are each independently
H, a C.sub.1 to C.sub.17 linear or branched alkyl chain, m is 0 to
17 and n is 0 to 2; or any omega amino aliphatic, terminal aryl or
aralkyl, including groups such as methyl, dimethyl, ethyl, propyl,
isopropyl, butyl, isobutyl, pentyl, hexyl, allyl, cyclopropane
methyl, hexanoyl, heptanoyl, acetyl, propionoyl, butanoyl,
phenylacetyl, cyclohexylacetyl, naphthylacetyl, cinnamoyl, phenyl,
benzyl, benzoyl, 12-Ado, 7'-amino heptanoyl, 6-Ahx, Amc or 8-Aoc,
or any single natural or unnatural .alpha.-amino acid, .beta.-amino
acid or .alpha., .alpha.-disubstituted amino acid, including all
(R) or (S) configurations of the foregoing, optionally in
combination with any of the foregoing non-amino acid capping
groups. In the foregoing, it is to be understood that, for example,
--C(.dbd.O)--NH--(CH.sub.2).sub.m--NH--C(.dbd.O)--CH(N(v.sub.1)(v.sub.2))-
((CH.sub.2).sub.m--N(v.sub.1)(v.sub.2)) is:
##STR00013##
[0191] The term "N-terminus capping group" includes any terminal
group attached through the terminal amine of the N-terminus of a
construct. The terminal amine may form a part of a residue, or may
form a part of an amino acid surrogate. In a preferred aspect, the
N-terminus capping group forms a part of an amino acid surrogate
which is at the N-terminus position of the construct. The
N-terminus capping group includes, but is not limited to, any omega
amino aliphatic, acyl group or terminal aryl or aralkyl including
groups such as methyl, dimethyl, ethyl, propyl, isopropyl, butyl,
isobutyl, pentyl, hexyl, allyl, cyclopropane methyl, hexanoyl,
heptanoyl, acetyl, propionoyl, butanoyl, phenylacetyl,
cyclohexylacetyl, naphthylacetyl, cinnamoyl, phenyl, benzyl,
benzoyl, 12-Ado, 7-amino heptanoyl, 6-Ahx, Amc or 8-Aoc, or
alternatively an N-terminus capping group is
--(CH.sub.2).sub.m--NH(v.sub.3), --(CH.sub.2).sub.m--CH.sub.3,
--C(.dbd.O)--(CH.sub.2).sub.m--CH.sub.3,
--C(.dbd.O)--(CH.sub.2).sub.m--NH(v.sub.3),
--C(.dbd.O)--(CH.sub.2).sub.m--C(.dbd.O)--OH,
--C(.dbd.O)--(CH.sub.2).sub.m--C(.dbd.O)-(v.sub.4),
--(CH.sub.2).sub.m--C(.dbd.O)--OH,
--(CH.sub.2).sub.m--C(.dbd.O)-(v.sub.4),
C(.dbd.O)--(CH.sub.2).sub.m--O(v.sub.3),
--(CH.sub.2).sub.m--O(v.sub.3),
C(.dbd.O)--(CH.sub.2).sub.m--S(v.sub.3), or
--(CH.sub.2).sub.m--S(v.sub.3), where v.sub.3 is H or a C.sub.1 to
C.sub.17 linear or branched alkyl chain, and v.sub.4 is a C.sub.1
to C.sub.17 linear or branched alkyl chain and m is 0 to 17.
[0192] A phenyl ring is "substituted" when the phenyl ring includes
one or more substituents independently comprising hydroxyl,
halogen, alkyl, or aryl groups attached directly or through an
ether linkage. Where the phenyl ring is so substituted, the amino
acid residue may be referred to as substituted, as in substituted
Phe, substituted HPhe or substituted Pgl.
[0193] The term "alkene" includes unsaturated hydrocarbons that
contain one or more double carbon-carbon bonds. Examples of alkene
groups include ethylene, propene, and the like.
[0194] The term "alkenyl" includes a linear monovalent hydrocarbon
radical of two to six carbon atoms or a branched monovalent
hydrocarbon radical of three to six carbon atoms containing at
least one double bond; examples thereof include ethenyl,
2-propenyl, and the like.
[0195] The "alkyl" groups specified herein include those alkyl
radicals of the designated length in either a straight or branched
configuration. Examples of alkyl radicals include methyl, ethyl,
propyl, isopropyl, butyl, sec-butyl, tertiary butyl, pentyl,
isopentyl, hexyl, isohexyl, and the like.
[0196] The term "alkynyl" includes a linear monovalent hydrocarbon
radical of two to six carbon atoms or a branched monovalent
hydrocarbon radical of three to six carbon atoms containing at
least one triple bond; examples thereof include ethynyl, propynal,
butynyl, and the like.
[0197] The term "aryl" includes a monocyclic or bicyclic aromatic
hydrocarbon radical of 6 to 12 ring atoms, and optionally
substituted independently with one or more substituents selected
from alkyl, haloalkyl, cycloalkyl, alkoxy, alkylthio, halo, nitro,
acyl, cyano, amino, monosubstituted amino, disubstituted amino,
hydroxy, carboxy, or alkoxy-carbonyl. Examples of aryl groups
include phenyl, biphenyl, naphthyl, 1-naphthyl, and 2-naphthyl,
derivatives thereof, and the like.
[0198] The term "aralkyl" includes a radical --R.sup.aR.sup.b where
R.sup.a is an alkylene (a bivalent alkyl) group and R.sup.b is an
aryl group as defined above. Examples of aralkyl groups include
benzyl, phenylethyl, 3-(3-chlorophenyl)-2-methylpentyl, and the
like.
[0199] The term "aliphatic" includes compounds with hydrocarbon
chains, such as for example alkanes, alkenes, alkynes, and
derivatives thereof.
[0200] The term "acyl" includes a group R--C(.dbd.O)--, where R is
an organic group. An example is the acetyl group
CH.sub.3--C(.dbd.O)--, referred to herein as "Ac".
[0201] A peptide or aliphatic moiety is "acylated" when an aryl,
alkyl or substituted alkyl group as defined above is bonded through
one or more carbonyl {--(C.dbd.O)--} groups. A peptide is most
usually acylated at the N-terminus.
[0202] An "omega amino aliphatic" includes an aliphatic moiety with
a terminal amino group. Examples of omega amino aliphatics include
7'-amino-heptanoyl and the amino acid side chain moieties of
ornithine and lysine.
[0203] The term "heteroaryl" includes mono- and bicyclic aromatic
rings containing from 1 to 4 heteroatoms selected from nitrogen,
oxygen and sulfur. 5- or 6-membered heteroaryl are monocyclic
heteroaromatic rings; examples thereof include thiazole, oxazole,
thiophene, furan, pyrrole, imidazole, isoxazole, pyrazole,
triazole, thiadiazole, tetrazole, oxadiazole, pyridine, pyridazine,
pyrimidine, pyrazine, and the like. Bicyclic heteroaromatic rings
include, but are not limited to, benzothiadiazole, indole,
benzothiophene, benzofuran, benzimidazole, benzisoxazole,
benzothiazole, quinoline, benzotriazole, benzoxazole, isoquinoline,
purine, furopyridine and thienopyridine.
[0204] An "amide" includes compounds that have a trivalent nitrogen
attached to a carbonyl group (--C(.dbd.O)--NH.sub.2), such as for
example methylamide, ethylamide, propylamide, and the like.
[0205] An "imide" includes compounds containing an imido group
(--C(.dbd.O)--NH--C(.dbd.O)--).
[0206] An "amine" includes compounds that contain an amino group
(--NH.sub.2).
[0207] A "nitrile" includes compounds that are carboxylic acid
derivatives and contain a (--CN) group bound to an organic
group.
[0208] The term "halogen" is intended to include the halogen atoms
fluorine, chlorine, bromine and iodine, and groups including one or
more halogen atoms, such as --CF.sub.3 and the like.
[0209] The term "composition", as in pharmaceutical composition, is
intended to encompass a product comprising the active
ingredient(s), and the inert ingredient(s) that make up the
carrier, as well as any product which results, directly or
indirectly, from combination, complexation or aggregation of any
two or more of the ingredients, or from dissociation of one or more
of the ingredients, or from other types of reactions or
interactions of one or more of the ingredients. Accordingly, the
pharmaceutical compositions of the present invention encompass any
composition made by admixing a construct of the present invention
and a pharmaceutically acceptable carrier.
[0210] The term "EC.sub.50" is intended to include the molar
concentration of an agonist which produced 50% of the maximum
possible response for that agonist. By way of example, a construct
which, at a concentration of 72 nM, produces 50% of the maximum
possible response for that construct as determined in a cGMP assay,
has an EC.sub.50 of 72 nM. Unless otherwise specified, the molar
concentration associated with an EC.sub.50 determination is in
nanomoles (nM).
[0211] The term "Ki (nM)" is intended to include the equilibrium
receptor binding affinity representing the molar concentration of a
competing compound that binds to half the binding sites of a
receptor at equilibrium in the absence of a competitor. In general,
the Ki is inversely correlated to the affinity of the compound for
the receptor, such that if the Ki is low, the affinity is high. Ki
may be determined using the equation of Cheng and Prusoff (Cheng
Y., Prusoff W. H., Biochem. Pharmacol. 22: 3099-3108, 1973):
Ki = EC 50 1 + [ ligand ] K d ##EQU00001##
where "ligand" is the concentration of ligand, which may be a
radioligand, and K.sub.d is an inverse measure of receptor affinity
which produces 50% receptor occupancy. Unless otherwise specified,
the molar concentration associated with a Ki determination is
nM.
[0212] The chemical naming protocol and structure diagrams used
herein employ and rely on the chemical naming features as utilized
by the ChemDraw program (available from Cambridgesoft Corp.,
Cambridge, Mass.). In particular, certain compound names were
derived from the structures using the Autonom program as utilized
by Chemdraw Ultra or ISIS base (MDL Corp.). In general, structure
diagrams do not depict hydrogen atoms associated with carbon atoms
other than in terminal groups and other special circumstances.
[0213] Certain structure diagrams and drawings herein, such as
those included in Tables 1 and 2, depict constructs composed of
amino acid surrogates and amino acid residues, with the surrogates
identified by structure diagrams and the amino acid residues
identified by a three letter abbreviation. Unless otherwise
specified, it is understood that the bond between the surrogate and
residue, or between the residue and surrogate, or between a
surrogate and residues on both the N-terminus and C-terminus side
thereof, is a conventional peptide bond, --C(.dbd.O)--NH-- or, in
the case where the peptide bond is to the ring nitrogen on the
N-terminus of the surrogate, --C(.dbd.O)--N--. In general, in the
depiction of such bonds the atoms of the amino acid surrogate are
depicted (e.g., --C(.dbd.O)-- or --N), but atoms of the amino acid
residue are not depicted.
Formulation and Utility
[0214] The constructs disclosed herein can be used for both medical
applications and animal husbandry or veterinary applications.
Typically, the construct, or a pharmaceutical composition including
the construct, is used in humans, but may also be used in other
mammals. The term "patient" is intended to denote a mammalian
individual, and is so used throughout the specification and in the
claims. The primary applications of this invention involve human
patients, but this invention may be applied to laboratory, farm,
zoo, wildlife, pet, sport or other animals.
[0215] The constructs disclosed herein may be used for the
treatment of any condition, syndrome or disease for which induction
of anti-hypertensive, cardiovascular, renal, and/or endocrine
effects are desired. This includes specifically any condition,
syndrome or disease for which a native natriuretic peptide may be
employed. Thus the constructs disclosed herein may be employed to
cause desired natriuresis, diuresis and/or vasodilation in a
patient.
[0216] In one aspect, the constructs disclosed herein are used in
the treatment of early stage, such as class 1, congestive heart
failure. In another aspect, the constructs disclosed herein are
used in the treatment of chronic or decompensated congestive heart
failure. In another aspect, the constructs disclosed herein are
used in the treatment of acute congestive heart failure, including
acutely decompensated congestive heart failure of patients with
dyspnea at rest or with minimal activity.
[0217] Salt Form of Constructs. The constructs of this invention
may be in the form of any pharmaceutically acceptable salt. The
term "pharmaceutically acceptable salts" refers to salts prepared
from pharmaceutically acceptable non-toxic bases or acids including
inorganic or organic bases and inorganic or organic acids. Salts
derived from inorganic bases include salts of aluminum, ammonium,
calcium, copper, ferric, ferrous, lithium, magnesium, manganic,
manganous, potassium, sodium, zinc, and the like. Particularly
preferred are the ammonium, calcium, lithium, magnesium, potassium,
and sodium salts. Salts derived from pharmaceutically acceptable
organic non-toxic bases include salts of primary, secondary, and
tertiary amines, substituted amines including naturally occurring
substituted amines, cyclic amines, and basic ion exchange resins,
such as arginine, betaine, caffeine, choline,
N,N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol,
2-dimethylaminoethanol, ethanolamine, ethylenediamine,
N-ethyl-morpholine, N-ethylpiperidine, glucamine, glucosamine,
histidine, hydrabamine, isopropylamine, lysine, methylglucamine,
morpholine, piperazine, piperidine, polyamine resins, procaine,
purines, theobromine, triethylamine, trimethylamine,
tripropylamine, tromethamine, and the like.
[0218] When the construct of the present invention is basic, acid
addition salts may be prepared from pharmaceutically acceptable
non-toxic acids, including inorganic and organic acids. Such acids
include acetic, benzenesulfonic, benzoic, camphorsulfonic,
carboxylic, citric, ethanesulfonic, formic, fumaric, gluconic,
glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic,
malic, mandelic, methanesulfonic, malonic, mucic, nitric, pamoic,
pantothenic, phosphoric, propionic, succinic, sulfuric, tartaric,
p-toluenesulfonic acid, trifluoroacetic acid, and the like. Acid
addition salts of the constructs of this invention are prepared in
a suitable solvent from the construct and an excess of an acid,
such as hydrochloric, hydrobromic, sulfuric, phosphoric, acetic,
trifluoroacetic, citric, tartaric, maleic, succinic or
methanesulfonic acid. The acetate salt form is especially useful.
Where the constructs of embodiments of this invention include an
acidic moiety, suitable pharmaceutically acceptable salts may
include alkali metal salts, such as sodium or potassium salts, or
alkaline earth metal salts, such as calcium or magnesium salts.
[0219] In addition, Applicants have advantageously discovered that
certain salt forms of the peptide constructs of the invention,
including pamoate, octanoate, decanoate, oleate, stearate, sodium
tannate and palmitate salt forms, have an increased circulation
half-life, in some cases doubled, versus the corresponding acetate
salt form. These salt forms are particularly well-suited for
administration by subcutaneous injection or intramuscular
injection, especially for chronic treatment, due to the reduced
frequency of dosing that may be achieved as a result of the longer
half-lives. While not being limited by theory, it is believed the
increased half-life is related to a decrease in solubility in
comparison to the acetate salt form. The increased half-life salt
forms of the peptide constructs of the invention may be
manufactured by any method including, for example, ion exchange,
mixing a solution of an acetate salt form of a construct with
disodium pamoate to form a pamoate suspension, or use of the
desired salt during the final purification step(s) in the
manufacture of the constructs.
[0220] Pharmaceutical Compositions. Another embodiment of the
present invention provides a pharmaceutical composition that
includes a construct of this invention and a pharmaceutically
acceptable carrier. The carrier may be a liquid formulation, and is
preferably a buffered, isotonic, aqueous solution. Pharmaceutically
acceptable carriers also include excipients, such as diluents,
carriers and the like, and additives, such as stabilizing agents,
preservatives, solubilizing agents, buffers and the like, as
hereafter described.
[0221] The constructs of the several embodiments of the present
invention may be formulated or compounded into pharmaceutical
compositions that include at least one construct of this invention
together with one or more pharmaceutically acceptable carriers,
including excipients, such as diluents, carriers and the like, and
additives, such as stabilizing agents, preservatives, solubilizing
agents, buffers and the like, as may be desired. Formulation
excipients may include polyvinylpyrrolidone, gelatin, hydroxy
cellulose, acacia, polyethylene glycol, manniton, sodium chloride
and sodium citrate. For injection or other liquid administration
formulations, water containing at least one or more buffering
constituents is preferred, and stabilizing agents, preservatives
and solubilizing agents may also be employed. For solid
administration formulations, any of a variety of thickening,
filler, bulking and carrier additives may be employed, such as
starches, sugars, fatty acids and the like. For topical
administration formulations, any of a variety of creams, ointments,
gels, lotions and the like may be employed. For most pharmaceutical
formulations, non-active ingredients will constitute the greater
part, by weight or volume, of the preparation. For pharmaceutical
formulations, it is also contemplated that any of a variety of
measured-release, slow-release or time-release formulations and
additives may be employed, so that the dosage may be formulated so
as to effect delivery of a construct of this invention over a
period of time. For example, gelatin, sodium carboxymethylcellulose
and/or other cellulosic excipients may be included to provide
time-release or slower-release formulations, especially for
administration by subcutaneous and intramuscular injection.
[0222] In general, the actual quantity of constructs administered
to a patient will vary between fairly wide ranges depending on the
mode of administration, the formulation used, and the response
desired.
[0223] In practical use, the constructs can be combined as the
active ingredient in an admixture with a pharmaceutical carrier
according to conventional pharmaceutical compounding techniques.
The carrier may take a wide variety of forms depending on the form
of preparation desired for administration, for example, oral,
parenteral (including intravenous), urethral, vaginal, nasal,
dermal, transdermal, pulmonary, deep lung, inhalation, buccal,
sublingual, or the like. In preparing the compositions for oral
dosage form, any of the usual pharmaceutical media may be employed,
such as, for example, water, glycols, oils, alcohols, flavoring
agents, preservatives, coloring agents and the like in the case of
oral liquid preparations, such as, for example, suspensions,
elixirs and solutions; or carriers such as starches, sugars,
microcrystalline cellulose, diluents, granulating agents,
lubricants, binders, disintegrating agents and the like in the case
of oral solid preparations such as, for example, powders, hard and
soft capsules and tablets.
[0224] Because of their ease of administration, tablets and
capsules represent an advantageous oral dosage unit form. If
desired, a composition including a construct of this invention may
be coated by standard aqueous or nonaqueous techniques. The amount
of active construct in such therapeutically useful compositions is
such that an effective dosage will be obtained. In another
advantageous dosage unit form, sublingual pharmaceutical
compositions may be employed, such as sheets, wafers, tablets or
the like. The active construct can also be administered
intranasally as, for example, by liquid drops or spray.
[0225] The tablets, pills, capsules, and the like may also contain
a binder such as gum tragacanth, acacia, corn starch or gelatin;
excipients such as dicalcium phosphate; a disintegrating agent such
as corn starch, potato starch or alginic acid; a lubricant such as
magnesium stearate; and a sweetening agent such as sucrose, lactose
or saccharin. When a dosage unit form is a capsule, it may contain,
in addition to materials of the above type, a liquid carrier such
as a fatty oil.
[0226] Various other materials may be utilized as coatings or to
modify the physical form of the dosage unit. For instance, tablets
may be coated with shellac, sugar or both. A syrup or elixir may
contain, in addition to the active ingredient, sucrose as a
sweetening agent, methyl and propylparabens as preservatives, a dye
and a flavoring such as cherry or orange flavor.
[0227] Constructs may also be administered parenterally. Solutions
or suspensions of these active peptides may be prepared in water
suitably mixed with a surfactant such as hydroxy-propylcellulose.
Dispersions may also be prepared in glycerol, liquid polyethylene
glycols and mixtures thereof in oils. These preparations may
optionally contain a preservative to prevent the growth of
microorganisms. Lyophilized single unit formulations may also be
utilized, which are reconstituted, such as with saline, immediately
prior to administration, and thus do not require a
preservative.
[0228] The pharmaceutical forms suitable for injectable use
include, for example, sterile aqueous solutions or dispersions and
sterile powders, such as lyophilized formulations, for the
extemporaneous preparation of sterile injectable solutions or
dispersions. In all cases, the form must be sterile and must be
fluid to the extent that it may be administered by syringe. The
form must be stable under the conditions of manufacture and storage
and must be preserved against the contaminating action of
microorganisms such as bacteria and fungi. The carrier can be a
solvent or dispersion medium containing, for example, water,
ethanol, a polyol, for example glycerol, propylene glycol or liquid
polyethylene glycol, suitable mixtures thereof, and vegetable
oils.
[0229] Constructs as disclosed herein may be therapeutically
applied by means of nasal administration. By "nasal administration"
is meant any form of intranasal administration of any of the
constructs of this invention. The constructs may be in an aqueous
solution, such as a solution including saline, citrate or other
common excipients or preservatives. The constructs may also be in a
dry or powder formulation.
[0230] In an alternative embodiment, constructs may be administered
directly into the lung. Intrapulmonary administration may be
performed by means of a metered dose inhaler, a device allowing
self-administration of a metered bolus of a construct of this
invention when actuated by a patient during inspiration. Both dry
powder inhalation and nebulized aerosols may be employed.
[0231] According to another embodiment of the present invention,
constructs of this invention may be formulated with any of a
variety of agents that increase effective nasal absorption of
drugs, including peptide drugs. These agents should increase nasal
absorption without unacceptable damage to the mucosal membrane.
U.S. Pat. Nos. 5,693,608, 5,977,070 and 5,908,825, among others,
teach a number of pharmaceutical compositions that may be employed,
including absorption enhancers, and the teachings of each of the
foregoing, and all references and patents cited therein, are
incorporated by reference.
[0232] If in an aqueous solution, certain constructs of the present
invention may be appropriately buffered by means of saline,
acetate, phosphate, citrate, acetate or other buffering agents,
which may be at any physiologically acceptable pH, generally from
about pH 4 to about pH 7. A combination of buffering agents may
also be employed, such as phosphate buffered saline, a saline and
acetate buffer, and the like. In the case of saline, a 0.9% saline
solution may be employed. In the case of acetate, phosphate,
citrate, acetate and the like, a 50 mM solution may be employed. In
addition to buffering agents, a suitable preservative may be
employed, to prevent or limit bacteria and other microbial growth.
One such preservative that may be employed is 0.05% benzalkonium
chloride.
[0233] It is also possible and contemplated that the construct may
be in a dried and particulate form. In a preferred embodiment, the
particles are between about 0.5 and 6.0 .mu.m, such that the
particles have sufficient mass to settle on the lung surface, and
not be exhaled, but are small enough that they are not deposited on
surfaces of the air passages prior to reaching the lung. Any of a
variety of different techniques may be used to make dry powder
microparticles, including but not limited to micro-milling, spray
drying and a quick freeze aerosol followed by lyophilization. With
micro-particles, the constructs may be deposited to the deep lung,
thereby providing quick and efficient absorption into the
bloodstream. Further, with such approach penetration enhancers are
not required, as is sometimes the case in transdermal, nasal or
oral mucosal delivery routes. Any of a variety of inhalers can be
employed, including propellant-based aerosols, nebulizers, single
dose dry powder inhalers and multidose dry powder inhalers. Common
devices in current use include metered dose inhalers, which are
used to deliver medications for the treatment of asthma, chronic
obstructive pulmonary disease and the like. Preferred devices
include dry powder inhalers, designed to form a cloud or aerosol of
fine powder with a particle size that is always less than about 6.0
.mu.m.
[0234] Microparticle size, including mean size distribution, may be
controlled by means of the method of making. For micro-milling, the
size of the milling head, speed of the rotor, time of processing
and the like control the microparticle size. For spray drying, the
nozzle size, flow rate, dryer heat and the like control the
microparticle size. For making by means of quick freeze aerosol
followed by lyophilization, the nozzle size, flow rate,
concentration of aerosoled solution and the like control the
microparticle size. These parameters and others may be employed to
control the microparticle size.
[0235] The constructs of this invention may be therapeutically
administered by means of an injection, typically a deep
intramuscular injection, such as in the gluteal or deltoid muscle,
of a time release injectable formulation. In one embodiment, a
construct of this invention is formulated with a PEG, such as
poly(ethylene glycol) 3350, and optionally one or more additional
excipients and preservatives, including but not limited to
excipients such as salts, polysorbate 80, sodium hydroxide or
hydrochloric acid to adjust pH, and the like. In another
embodiment, a construct of this invention is formulated with a
poly(ortho ester), which may be an auto-catalyzed poly(ortho ester)
with any of a variable percentage of lactic acid in the polymeric
backbone, and optionally one or more additional excipients. In one
embodiment poly (D,L-lactide-co-glycolide) polymer (PLGA polymer)
is employed, preferably a PLGA polymer with a hydrophilic end
group, such as PLGA RG502H from Boehringer Ingelheim, Inc.
(Ingelheim, Germany). Such formulations may be made, for example,
by combining a construct of this invention in a suitable solvent,
such as methanol, with a solution of PLGA in methylene chloride,
and adding thereto a continuous phase solution of polyvinyl alcohol
under suitable mixing conditions in a reactor. In general, any of a
number of injectable and biodegradable polymers, which are
preferably also adhesive polymers, may be employed in a time
release injectable formulation. The teachings of U.S. Pat. Nos.
4,938,763, 6,432,438, and 6,673,767, and the biodegradable polymers
and methods of formulation disclosed therein, are incorporated here
by reference. The formulation may be such that an injection is
required on a weekly, monthly or other periodic basis, depending on
the concentration and amount of construct, the biodegradation rate
of the polymer, and other factors known to those of skill in the
art.
[0236] Routes of Administration. If it is administered by
injection, the injection may be intravenous, subcutaneous,
intramuscular, intraperitoneal or other means known in the art. The
constructs of this invention may be formulated by any means known
in the art, including but not limited to formulation as tablets,
capsules, caplets, suspensions, powders, lyophilized preparations,
suppositories, ocular drops, skin patches, oral soluble
formulations, sprays, aerosols and the like, and may be mixed and
formulated with buffers, binders, excipients, stabilizers,
anti-oxidants and other agents known in the art. In general, any
route of administration by which the constructs of this invention
are introduced across an epidermal layer of cells may be employed.
Administration means may thus include administration through mucous
membranes, buccal administration, oral administration, dermal
administration, inhalation administration, pulmonary
administration, nasal administration, urethral administration,
vaginal administration, and the like.
[0237] In one aspect, a construct of this invention is administered
by means of a time-release injectable formulation, such as a
construct of this invention in a formulation with a PEG, poly(ortho
ester) or PLGA polymer. In another aspect, a construct of this
invention is administered by means of an automated delivery device
providing subcutaneous delivery, either continuous or intermittent.
Any of the foregoing methods and formulations are particularly
applicable for treatment of chronic conditions or syndromes,
including chronic congestive heart failure and particularly chronic
decompensated congestive heart failure.
[0238] In one aspect, any construct of this invention may be
administered by subcutaneous administration, including all the
methods disclosed in U.S. Pat. No. 6,586,396. In another aspect, a
patient, particularly a patient who is relatively compensated or is
a patient with congestive heart failure in an outpatient setting,
may be administered a construct of this invention by methods and in
doses as disclosed in U.S. Patent Application Publication
2004/0077537 and International Patent Application Publication WO
2003/079979. In another aspect, a patient may be administered a
construct of this invention by means of the methods as disclosed in
U.S. Patent Application Publication 2005/0113286. In yet another
aspect, a patient who has undergone myocardial injury may be
treated for cardiac remodeling by means of the methods as disclosed
in U.S. Patent Application Publication 2006/0019890.
[0239] A construct of this invention may also be administered by
transdermal administration, including by means of the delivery
system, including the apparatus, and the methods as disclosed in
U.S. Patent Application Publication 2006/0034903. Similarly, the
hydrogel formulations and solid state formulations disclosed
therein may be adapted for use with the constructs of this
invention.
[0240] Therapeutically Effective Amount. In general, the actual
quantity of a construct of this invention administered to a patient
will vary between fairly wide ranges depending upon the mode of
administration, the formulation used, and the response desired. The
dosage for treatment is administration, by any of the foregoing
means or any other means known in the art, of an amount sufficient
to bring about the desired therapeutic effect. Thus, a
therapeutically effective amount includes an amount of a construct
or pharmaceutical composition of this invention that is sufficient
to induce a desired effect, including specifically an
anti-hypertensive, cardiovascular, renal and/or endocrine effect.
In one aspect a therapeutically effective amount is an amount that
results in desired natriuresis, diuresis and/or vasodilation.
[0241] In general, the constructs of this invention are highly
active. For example, a construct can be administered at about
0.001, 0.01, 0.05, 0.1, 0.5, 1, 5, 10 or 100 .mu.g/kg body weight,
depending on the specific construct selected, the desired
therapeutic response, the route of administration, the formulation
and other factors known to those of skill in the art.
Combination Therapy
[0242] It is also possible and contemplated that constructs
according to several embodiments of the present invention are used
in combination with other drugs or agents, particularly in the
treatment of congestive heart failure.
[0243] According to another aspect of the present invention, a
method for treating congestive heart failure is provided. The
method includes administering to the patient having congestive
heart failure a therapeutically effective amount of a construct as
disclosed herein in combination with a therapeutically effective
amount of another compound that is useful in the treatment of
congestive heart failure, or alternative that is useful in
extending the bioavailability of a construct of this invention in a
patient. In one aspect, a patient may be administered a construct
of this invention in combination with a diuretic, such as by means
of the methods and diuretics disclosed in U.S. Patent Application
Publication 2004/0063630. Diuretics which may be employed in
combination include thiazide-, loop- and potassium-sparing
diuretics, including without limitation diuretics such as
hydrochlorothiazide (Hydrodiuril.RTM.), chlorthalindone, furosemide
(Lasix.RTM.), spironolactone (Aldactone.RTM.) and triamterine.
[0244] In another aspect of the present invention, a method for
treating congestive heart failure is provided by administering a
therapeutically effective amount of a construct as disclosed herein
in combination with a therapeutically effective amount of an
anti-hypertensive agent other than a diuretic. Such
anti-hypertensive agents include generally calcium channel blockers
(including dihydropyridines and non-dihydropyridines),
sympatholytic agents, non-specific adrenergic blocking agents,
.alpha.-adrenergic antagonists (including nonselective and
selective .alpha.1-blocking agents), 1-blockers (including
non-selective as well as selective blockers and those with
intrinsic sympathomimetic activity), vasodilators (for treatment of
resistant and emergent hypertension), angiotensin converting enzyme
inhibitors and angiotensin II antagonists. Anti-hypertensive agents
that may be employed in combination include mixed .alpha. and
.beta. antagonists such as labetolol (Normodyne.RTM.); vasodilators
such as hyralazine (Apresoline.RTM.), minoxidil (Loniten.RTM.),
nitroprusside (Nipride.RTM.), or diazoxide (Hyperstat IV.RTM.);
calcium blockers such as nifedipine (Adalat.RTM.), diltiazem
(Cardizem.RTM.), or verapamil (Calan.RTM.); sympatholytics such as
clonidine (Catapres.RTM.), methyldopa (Aldomet.RTM.), reserpine
(Serpasil.RTM.), or guanethidine (Ismelin.RTM.); ACE inhibitors
such as captopril (Capoten.RTM.), enalapril (Vasotec.RTM.) or
lisinopril (Prinivil.RTM.); .alpha.-adrenergic antagonists such as
phentolamine (Regitine.RTM.) or prazosin (Minipress.RTM.);
angiotensin II antagonists such as losartan (Cozaar.RTM.); or
.beta.-adrenergic antagonists such as propranolol (Inderal.RTM.),
nadolol (Corgard.RTM.), metoprolol (Lopressor.RTM.) or
pindolol.
Other Indications
[0245] While a primary use of the constructs of the present
invention is in the treatment and amelioration of symptoms of
congesive heart failure, acute kidney failure and renal
hypertension, the constructs of the present invention may be
employed in any treatment scheme or modality for which natriuretic,
diuretic and/or vasodilator compounds provide a therapeutic
benefit. Thus, in one aspect, the constructs of the present
invention may be employed as additives to peritoneal dialysis
solutions, as disclosed in U.S. Pat. No. 5,965,533. In another
aspect, the constructs of the present invention may be employed in
opthomologic applications, as disclosed in International Patent
Publication WO 00/18422.
Synthetic Methods of Amino Acid Surrogates
[0246] The following examples of methods of synthesis of amino acid
surrogates of the invention are intended to be exemplary, and it is
to be understood that variations thereon may be undertaken by one
of skill in the art, and such variations are intended to be
included herein.
Synthesis of Ketopiperazine Scaffolds Mimicking Amino Acids without
Functionalized R Side Chain (Methods A and B)
[0247] The constructs were prepared by a variety of methods as
described in Methods A and B.
[0248] Method A: The dipeptides (3) were formed by the mixed
anhydride method, using Boc-serine (OBn)-OH (1), and an
.alpha.-amino ester (2). The dipeptides were obtained in high
yields, and usually no purification was required. Reduction of both
the methyl ester and the amide group was done using
diborane-tetrahydrofuran, with the secondary amines protected to
give the di-Boc protected amino alcohol intermediates (4).
Oxidation of the alcohols with pyridinium dichromate (PDC) with
concomitant cyclization gave the piperazine-2-ones (5) in one step.
Benzyl ether removal by hydrogenation, followed by protecting group
exchange gave the Fmoc protected piperazine-2-ones (6). Finally,
the primary alcohol was oxidized to the acid by any of a number of
different methods (PDC, Jones oxidation, ruthenium chloride-sodium
periodate, 2,2,6,6-tetramethyl-1-piperidinyloxy, free radical
(TEMPO) oxidation) to give the final products (7).
##STR00014##
[0249] Synthesis of
2-(3-benzyloxy-2-tert-butoxycarbonylamino-propionylamino)-2-substituted
acetic acid methyl ester (3): To a solution of 10 mmol of Boc
serine benzyl ether (1) in 30 mL of dry tetrahydrofuran, kept at
-20.degree. C. under nitrogen, was added 22 mmol of triethylamine,
followed by the slow addition of 11.4 mmol of
isobutylchloroformate. A white solid precipitated out. The slurry
was stirred for 15 minutes, and then 11.1 mmol of .alpha.-amino
ester (2) was added in one portion. The slurry was stirred at
-20.degree. C. for 30 minutes, and then allowed to warm up to room
temperature, stirred for 2 hours, and then concentrated to dryness.
The mixture was then partitioned between 50 mL of ethyl acetate/30
mL of 1N hydrochloric acid solution. The layers were separated, and
the organic layer washed with 1.times.20 mL of 1N hydrochloric
acid, and 1.times.20 mL of saturated sodium bicarbonate solution,
dried over magnesium sulfate and concentrated. Compounds (3) were
usually obtained in yields above 90%, and no purification was
required.
TABLE-US-00001 R Analytical Data for Compounds (3) ##STR00015##
.sup.1H NMR .delta. (CDCl.sub.3): 1.43 (s, 9H, .sup.tBu), 3.0-3.18
(two sets of dd, 2H, CH.sub.2-Ph), 3.50-3.57 (t, 1H, CH.sub.2O),
3.68 (s, 3H, CH.sub.3O), 3.87-3.96 (br. d, 1H, CH.sub.2O),
4.23-4.33 (br. m, 1H, CHN), 4.45-4.57 (dd, 2H, CH.sub.2O),
4.80-4.88 (m, 1H, CHN), 5.30-5.37 (m, 1H, NH), 7.0-7.38 (a series
of m, 10H, Ph), yield = 96%, t.sub.R = 6.88 min, (M.sup.+ + 1) =
456.99 ##STR00016## .sup.1H NMR .delta. (CDCl.sub.3): 0.81-0.96 (a
series of m, 6H, CH.sub.3), 1.00-1.16 (m, 1H, CH.sub.2), 1.30-1.45
(m, 1H, CH.sub.2), 1.45 (s, 9H, .sup.tBu), 1.80-1.96 (m, 1H, CH),
3.54-3.64 (dd, 1H, CH.sub.2O), 3.70 (s, 3H, CH.sub.3O), 3.82-3.96
(dd, 1H, CH.sub.2O), 4.28-4.40 (m, 1H, CHN), 4.51-4.61 (m, and s,
3H, CH.sub.2O, and CHN), 5.51-5.61 (br. d, 1H, NH), 7.12-7.37 (br.
m, 5H, Ph), yield = quant., t.sub.R = 6.93 min, (M.sup.+ + 1) =
423.25 ##STR00017## .sup.1H NMR .delta. (CDCl.sub.3): 1.45 (s, 9H,
.sup.tBu), 3.73 (s, 3H, CH.sub.3O), 3.84-3.90 (m, 2H, CH.sub.2N),
4.01-4.17 (m, 2H, CH.sub.2O), 4.32-4.38 (br. m, 1H, CHN), 4.54-4.58
(d, 2H, CH.sub.2O), 5.46-5.57 (d, 1H, NH), 7.05-7.12 (br. m, 1H,
Ph), 7.24-7.40 (m, 4H, Ph), yield = quant., t.sub.R = 5.51 min,
(M.sup.+ + 1) = 367.07
[0250] Synthesis of
Di-Boc-2-substituted-(2-amino-3-benzyloxy-propyl-amino)-ethanol
(4): To a solution of 35 mmol of (3) in 50 mL of dry
tetrahydrofuran, kept at room temperature under nitrogen, was added
200 mL of 1N diborane solution in tetrahydrofuran. The solution was
stirred at room temperature overnight, and then slowly poured over
an ice-cold solution of 200 mL of 1N hydrochloric acid solution.
The biphasic solution was then neutralized with solid sodium
hydroxide. 140 mL of a saturated solution of sodium bicarbonate was
added, followed by 70 mmol of di-tert-butyl-dicarbonate, and the
mixture stirred for 2 days at room temperature, diluted with 200 mL
of ethyl acetate and the layers separated. The organic layer was
dried over magnesium sulfate, and concentrated. The products (4)
were purified by silica gel column chromatography.
TABLE-US-00002 R Analytical Data for Compounds (4) ##STR00018##
.sup.1H NMR .delta. (CDCl.sub.3): 1.42 (s, 9H, .sup.tBu), 1.48 (s,
9H, .sup.tBu), 2.48-3.02 (a series of m, 2H, CH.sub.2-Ph), 3.1-3.48
(br. m, 1H, CH.sub.2O), 3.25-3.48 (br. m, 2H, CH.sub.2N), 3.50-3.75
(m, 2H, CH.sub.2O), 3.80-3.97 (m, 2H, CH.sub.2O, and CHN), 4.25
(br. m, 1H, CHN), 4.45 (s 2H, CH.sub.2O), 4.9 (br. s, 1H, OH), 5.3
(br. s, 1H, NH), 7.1-7.4 (m, 10H, Ph), yield = 76%, t.sub.R = 8.04
min, (M.sup.+ + 1) = 515.25 ##STR00019## .sup.1H NMR .delta.
(CDCl.sub.3): 0.84-0.96 (m, CH, CH.sub.2, CH.sub.3), 1.42 (s, 9H,
.sup.tBu), 1.45 (s, 9H, .sup.tBu), 1.42-1.44 (m, 1H, CH), 2.88-3.11
(br. m, 2H, CH.sub.2N), 3.42-3.57 (m, 2H, CH.sub.2O), 3.62-4.10
(two m, 4H, CH.sub.2O, and CHN), 4.51 (s, 2H, CH.sub.2O), 7.27-7.38
(m, 5H, Ph), yield = 80%, t.sub.R = 8.19 min, (M.sup.++ 1) = 481.26
##STR00020## .sup.1H NMR .delta. (CDCl.sub.3): 1.35-1.43 (m, 18H,
.sup.tBu), 3.20-3.32 (m, 1H, CH.sub.2N), 3.55-3.84 (a series of m,
8H, CH.sub.2N, CH.sub.2O), 3.90-4.05 (m, 1H, CHN), 4.45 (s, 2H,
CH.sub.2O), 4.9-5.02 (m, 1H, NH), 7.2-7.35 (m, 5H, Ph), yield =
56%, t.sub.R = 6.40 min, (M.sup.+ + 1) = 425.21
[0251] Synthesis of
1,4-di-Boc-6-benzyloxymethyl-3-substituted-piperazin-2-one (5): A
solution of 70 mmol of (4), and 400 mmol of pyridinium dichromate
in 300 mL of dry dimethylformamide was stirred at 48.degree. C.
under nitrogen for 6 hours, cooled to room temperature, poured into
1500 mL of water, and extracted with 4.times.500 mL of ethyl ether.
The ethereal layers were combined, dried over magnesium sulfate,
and concentrated. The products (5) were purified by silica gel
column chromatography.
TABLE-US-00003 R Analytical Data for Compounds (5) ##STR00021##
.sup.1H NMR .delta. (CDCl.sub.3): 1.4 (s, 9H, .sup.tBu), 1.5 (s,
9H, .sup.tBu), 3.05- 3.58 (a series of m, CH.sub.2-Ph, and
CH.sub.2N), 4.1-4.32 (a series of m, 2H, CH.sub.2N), 4.47 (s, 2H,
CH.sub.2O), 4.78-4.86 (br. m, 1H, CHN), 7.12-7.42 (m, 10H, Ph),
yield = 42%, t.sub.R = 8.65 min, (M.sup.+ + 1) = 511.05.
##STR00022## .sup.1H NMR .delta. (CDCl.sub.3): 0.82-1.56 (four s,
and four m, 27H, .sup.tBu, CH, CH.sub.2, and CH.sub.3), 3.20-3.52
(a series of m, 2H, CH.sub.2N), 3.60-3.88 (a series of m, 2H,
CH.sub.2O), 4.20-4.60 (a series of m, one s, 4H, CH.sub.2O, CHN),
7.21-7.37 (m, 5H, Ph), yield = 24%, t.sub.R = 9.23 min, (M.sup.+ +
1) = 477.32.
[0252] Synthesis of
4-Fmoc-6-hydroxymethyl-3-substituted-piperazin-2-one (6): A
suspension of 19 mmol of (5) and 2 g of 10% palladium on carbon in
200 mL of ethanol was hydrogenated at room temperature and
atmospheric pressure overnight. The suspension was filtered through
celite, and concentrated. The residue was redissolved in 40 mL of
50% trifluoroacetic acid in dichloromethane. The solution was
stirred at room temperature for 2 hours, and then concentrated. The
residue was redissolved in 60 mL of tetrahydrofuran/40 mL of water,
and neutralized with solid sodium bicarbonate, followed by the
addition of 40 mmol of solid sodium bicarbonate, and 20 mmol of
Fmoc chloride. The mixture was then stirred at room temperature for
2 hours, diluted with 300 mL of ethyl acetate, and the layers
separated. The organic layer was dried over magnesium sulfate,
concentrated, and purified by silica gel column chromatography.
TABLE-US-00004 R Analytical Data for Compound (6) ##STR00023##
.sup.1H NMR .delta. (CDCl.sub.3): 2.15-2.32 (br. m, 1H,
CH.sub.2--Ph), 2.70- 2.81 (br. m, 1H, CH.sub.2-Ph), 3.0-3.32 (br.
m, 3H, CHN, and CH.sub.2N), 3.47-3.65 (br. m, 3H, CH.sub.2O, and
CHN), 3.95-4.22 (two m, 2H, CH, and CHN), 4.32-4.48 (br. m, 2H,
CH.sub.2O), 4.84-4.92 (br. m, 1H, NH), 6.73-6.83 (br. m, 1H, Ph),
6.92- 7.01 (br. m, 1H, Ph), 7.08-7.82 (a series of m, 11H, Ph, and
fulvene), yield = 65%, t.sub.R = 5.78 min, (M.sup.+ + 1) = 443.07.
##STR00024## .sup.1H NMR .delta. (CDCl.sub.3): 0.6-1.15 (br. peaks,
7H, CH.sub.2, and CH.sub.3), 1.20-1.42 (br. m, 1H, CH.sub.2),
1.72-2.02 (two br. peaks, 1H, CH), 2.74-2.86 (t, 1/2H, CHN),
2.74-3.74 (a series of br. peaks, 5H, CH.sub.2O, CH.sub.2N, and
CHN), 4.16-4.22 (br. m, 1H, CH), 4.52-4.74 (br. m, 2H, CH.sub.2O),
7.24-7.82 (a series of m, 8H, fulvene), yield = 34%, t.sub.R = 5.72
min, (M.sup.+ + 1) = 408.95 ##STR00025## .sup.1H NMR .delta.
(CDCl.sub.3): 0.73-1.00 (m, 7H, CH.sub.3), 2.2-2.3 (br. m, 0.5H,
CH), 2.74-4.62 (a series of br. peaks, 12H, CH.sub.2N, CH.sub.2O
and CHN), 3.68 (s, 3H, CH.sub.3O), 7.26-7.77 (m, 9H, fulvene),
yield = 45% (3 steps), t.sub.R = 5.34 min, (M.sup.+ + 1) =
394.93
[0253] Synthesis of
4-Fmoc-5-substituted-6-oxo-piperazine-2-carboxylic acid (7):
Compounds (7) were prepared by several methods.
[0254] (a) To a biphasic solution of 10 mmol of (6) in 180 mL of
acetonitrile, 180 mL of carbon tetrachloride, and 240 mL of water,
cooled to 0.degree. C., was added 112 mmol of solid sodium
periodate, followed by 340 mg of ruthenium chloride. The reaction
was allowed to warm up to room temperature, stirred for 2 hours,
and then filtered through celite. The layers were separated, and
the aqueous layer re-extracted with 2.times.75 mL of ethyl acetate.
The organic layers were combined, dried over magnesium sulfate, and
concentrated.
[0255] (b) A solution of 12 mmol of (6), and 59 mmol of PDC in 60
mL of dry dimethylformamide was stirred at 48.degree. C. under
nitrogen for .about.5 hours, cooled to room temperature, and poured
over 600 mL of water, and extracted with 3.times.200 mL of
dichloromethane. The organic layers were combined, dried over
magnesium sulfate, and concentrated.
[0256] (c) To a solution of 17 mmol of (6) in 350 mL of acetone
kept at room temperature was added 25 mL of Jones reagent (prepared
from 8.0 g of chromic acid, 17.4 mL of water, and 6.9 mL of
concentrated sulfuric acid). The mixture was stirred for 1 hour,
150 mL of isopropanol was added, and the mixture filtered through
celite. The celite was washed with ethyl acetate. The organic
layers were combined and concentrated. The residue was dissolved in
250 mL of ethyl acetate and washed with 2.times.50 mL of water,
dried over magnesium sulfate, and concentrated.
[0257] (d) To a solution of 81 mmol alcohol (6) in 810 mL of
acetonitrile kept at room temperature, was added phosphate buffer
solution (prepared with 7.2 g of sodium phosphate monobasic, and
14.3 g of sodium phosphate dibasic in 295 mL of water), followed by
the addition of 3.3 g (20.7 mmol) of TEMPO, and 18.6 g (164.4 mmol)
of sodium chlorite, and the biphasic solution placed in an oil bath
kept at 43.degree. C., and then a solution of 43.3 mL (25.9 mmol)
of sodium hypochlorite solution (prepared by mixing 19.3 mL of
10-13% sodium hypochlorite solution, and 24 mL of water) was added
slowly. The reaction was stirred at 43.degree. C. for 4 hours. The
solution was cooled to room temperature, and a solution of 200 mL
of 10% sodium hydrogen sulfite solution was added, stirred for 10
minutes, diluted with 500 mL of ethyl acetate, and the layers
separated. The organic layer was washed with 1.times.100 mL of
brine, 1.times.160 mL of 1N hydrochloric acid solution, dried over
sodium sulfate, and concentrated.
[0258] The products (7) were purified by silica gel column
chromatography.
TABLE-US-00005 R Analytical Data for Compounds (7) ##STR00026##
.sup.1H NMR .delta. (CDCl.sub.3): 2.36-2.45 (dd, 1H, CH.sub.2-Ph),
2.62-2.76 (m, 1/2H, CH.sub.2-Ph), 2.82-2.98 (m, 1/2H, CH.sub.2-Ph),
3.13-3.25 (m, 1H, CH.sub.2N), 3.98-4.64 (a series of m, 6H, CHN,
CH.sub.2O, CH.sub.2, and CH), 4.87 (br. m, 1/2H, NH), 6.85 (br. s,
1H, Ph), 7.0-7.40 (a series of m, 12H, Ph and fulvene), 9.18-9.40
(br. d, 1H, CO.sub.2H), t.sub.R = 5.91 min, (M.sup.+ + 1) = 457.37.
##STR00027## .sup.1H NMR .delta. (CDCl.sub.3): 0.64-1.02
(overlapping t, 6H, CH.sub.3), 1.02-1.68 (three br. m, 2H,
CH.sub.2), 1.96-2.16 (br. m, 1H, CH), 2.88-3.18 (m, 1H, CH.sub.2N),
3.85-4.12 (three m, 2H, CH.sub.2N, and CHN), 4.18-4.35 (m, 1H, CH),
4.55-4.72 (m, 2H, CH.sub.2), 4.75-4.86 (br. m, 1H, NH), 7.28-7.82
(a series of m, 8H, fulvene), 9.1-9.26 (two br. s, 1H, CO.sub.2H),
t.sub.R = 5.86 min, (M.sup.+ + 1) = 423.20. ##STR00028## .sup.1H
NMR .delta. (CDCl.sub.3): 0.62-1.03 (m, 7H, CH.sub.3), 1.90-2.05
(br. m, 1H, CH), 2.87-4.60 (a series of br. peaks, 8H, CH.sub.2N,
CH.sub.2O and CHN and CH), 7.29-7.80 (m, 9H, fulvene), yield = 61%,
t.sub.R = 5.52 min, (M.sup.+ + 1) = 409.11
[0259] Method B: Intermediates
Di-Boc-2-substituted-(2-amino-3-benzyloxy-propyl-amino)-ethanols
(4), prepared as described in method A, were oxidized to the acid
using TEMPO/isocyanuric acid reagent, and then esterified with
iodomethane to give fully protected reduced dipeptide analogs (8).
Deprotection of the Boc groups, and the benzyl ether, gave
3-substituted 5-hydroxymethyl-piperazin-2-ones, which were
protected as the Fmoc derivatives to give (6), which were oxidized
to the final product as described in method A.
##STR00029##
[0260] Synthesis of
Di-Boc-(2-amino-3-benzyloxy-propylamino)-2-substituted acetic acid
methyl ester (8): To a suspension of 76 mmol of (4) in 680 mL of
acetone, and 210 mL of a saturated sodium bicarbonate solution,
kept at 0.degree. C., was added 21 mmol of solid sodium bromide,
and 2.9 mmol of TEMPO, followed by the slow addition of 156 mmol of
trichloroisocyanuric acid. The reaction was stirred for 30 minutes
at 0.degree. C., and then at room temperature overnight, acidified
with a solution of 1N hydrochloric acid, and extracted with
2.times.300 mL of ethyl acetate. The organic layer was washed with
3.times.50 mL of 1N hydrochloric acid, dried over magnesium
sulfate, and concentrated. The residue was redissolved in 40 mL of
dry dimethylformamide and 95 mmol of solid sodium bicarbonate, and
112 mmol of iodomethane was added, and the mixture stirred at room
temperature under nitrogen until HPLC showed the reaction was
complete; the solution was then diluted with 200 mL of ethyl ether,
and washed with 2.times.100 mL of water, dried over magnesium
sulfate, and concentrated. The products (8) were purified by silica
gel column chromatography.
TABLE-US-00006 R Analytical Data for Compounds (8) ##STR00030##
.sup.1H NMR .delta. (CDCl.sub.3): 1.41 (s, 9H, .sup.tBu), 1.46 (s,
9H, .sup.tBu), 2.44-2.58 (d, 1/2H, CH.sub.2-Ph), 2.66-2.88 (d,
1/2H, CH.sub.2-Ph), 3.16-3.46 (three sets of m, 5H, CH.sub.2-Ph,
CH.sub.2N, and CH.sub.2O), 3.72 (s, 3H, CH.sub.3O), 3.75-4.05 (two
m, 1H, CHN), 4.42 (s, 2H, CH.sub.2O), 4.95-5.10 (d, 1/2H, NH),
5.30-5.38 (d, 1/2H, NH), 7.10-7.38 (m, 10H, Ph), yield = 62%,
t.sub.R = 7.75 min, (M.sup.+ + 1) = 529.03. ##STR00031## .sup.1H
NMR .delta. (CDCl.sub.3): 1.41 (s, 9H, .sup.tBu), 1.42 (s, 9H,
.sup.tBu), 3.30-3.60 (br. m, 4H, CH.sub.2N, CH.sub.2O), 3.70 (s,
3H, CH.sub.3O), 3.75-3.95 (m, 2H, CH.sub.2N), 4.51 (s, 2H,
CH.sub.2O), 5.0-5.08 (br. s, 1H, NH), 7.25-7.37 (m, 5H, Ph), yield
= 47% t.sub.R = 7.28 min, (M.sup.+ + 1) = 453.17.
[0261] Synthesis of
4-Fmoc-6-hydroxymethyl-3-substituted-piperazin-2-one (6): A
solution of 36 mmol of (8) in 40 mL of 50% trifluoroacetic acid in
dichloromethane was stirred at room temperature for 2 hours, and
then poured in 200 mL of saturated sodium bicarbonate solution. The
layers were separated, and the organic layer concentrated. The
residue was redissolved in 100 mL of ethyl acetate, and washed with
2.times.50 mL of water, dried over magnesium sulfate, and
concentrated. The residue was dissolved in 100 mL of ethanol, and 5
g of 10% palladium on carbon and 35 mL of a 1N hydrochloric acid
solution was added, and the mixture hydrogenated at room
temperature and atmospheric pressure until HPLC showed the reaction
was complete; the solution was then filtered through celite and
concentrated. The residue was redissolved in 80 mL of ethyl
acetate, 70 mmol of sodium bicarbonate in 30 mL of water was added,
and the mixture stirred at room temperature overnight. The ethyl
acetate was removed and 100 mL of tetrahydrofuran was added,
followed by 178 mmol of solid sodium bicarbonate and 43 mmol of
Fmoc chloride, and the mixture was stirred until HPLC showed it was
complete, diluted with 300 mL of ethyl acetate, and the layers
separated. The organic layer was washed with 2.times.50 mL of
water, dried over magnesium sulfate, and concentrated. The products
(6) were purified by silica gel column chromatography.
[0262] Synthesis of
4-Fmoc-5-substituted-6-oxo-piperazine-2-carboxylic acid (7):
Compounds (7) were prepared as described in method A.
General Common Synthetic Scheme for the Preparation of
Ketopiperazine Scaffolds Applicable to Compounds With or Without
Functionalized R sidechains (Methods C, E, F)
[0263] Method C: (2-Fmoc-amino-3-R'--O-propylamino)-2-substituted
acetic acid methyl esters (10) were prepared by reductive amination
of Fmoc O-protected serinal (9) with .alpha.-amino esters (2),
using either sodium cyanoborohydride or sodium
triacetoxyborohydride as the reducing agent. The Fmoc O-protected
serinal (9) required for the reductive amination was prepared
according to method D, either by reduction of the ester (12) by
di-isobutylaluminun hydride, by oxidation of Fmoc O-protected
serinol (13) with Dess-Martin periodinane, or by reduction of the
Fmoc O-protected serine Weinreb amide (14) with lithium aluminum
hydride. The preferred method for the preparation of Fmoc
O-protected serinals (9) was the reduction of the Weinreb amide
analog. (2-Fmoc-amino-3-R'--O-propylamino)-2-substituted acetic
acid methyl esters (10) were then N and O deprotected, cyclized,
and Fmoc protected to give 3-substituted
6-hydroxymethyl-piperazin-2-ones (6), which were then oxidized to
the final product as described in method A.
[0264] The protecting group (R') on the hydroxyl group of
Fmoc-O-protected serinal (9) used in the synthesis depends on the
nature of the side chain R of the amino ester. When R contained no
functional groups, the side chain of Fmoc serine was protected as
the .sup.tBu ether. When R contained functional groups, the side
chain of Fmoc serine was protected as the trityl ether.
##STR00032##
##STR00033##
[0265] Method D: Synthesis of various Fmoc-O-protected serinals
(9). Synthesis of Fmoc-O--R' serine methyl ester (12): A slight
suspension of 80 mmol of Fmoc O--R' serine (11), 10.0 g (120 mmol)
of solid sodium bicarbonate, and 10.0 mL (160 mmol) of iodomethane
in 80 mL of dry dimethylformamide, kept under nitrogen, was stirred
at room temperature overnight. The reaction mixture was then poured
over 500 mL of water, and the solid filtered. The solid was
redissolved in 800 mL of ethyl acetate, and washed with 1.times.200
mL of water, dried over magnesium sulfate, and concentrated. No
purification was required.
TABLE-US-00007 R' Analytical Data for Compounds (12) .sup.tBu
.sup.1H NMR .delta. (CDCl.sub.3): 1.14 (s, 9H, .sup.tBu), 3.57-3.70
(m, 1H, CH.sub.2--O), 3.75 (s, 3H, O--CH.sub.3), 3.79-3.83 (m, 1H,
CH.sub.2--O), 4.01-4.50 (a series of multiples, 4H), 5.64-5.68 (d,
1H, NH), 7.28-7.78 (8H, fulvene), yield = 93% t.sub.R = 7.8 min.
Trt .sup.1H NMR .delta. (CDCl.sub.3): 3.42-3.48 (m, 1H,
CH.sub.2--O), 3.59-3.66 (m, 1H, CH.sub.2--O), 3.81 (s, 3H,
CH.sub.3--O), 4.10-4.18 (m, 1H, CH), 4.36-4.42 (m, 2H,
CH.sub.2--O), 4.50-4.57 (m, 1H, CH--N), 5.73-5.78 (d, 1H, NH),
7.22-7.82 (8H, fulvene), yield = quant., t.sub.R = 9.04 min.
[0266] Synthesis of Fmoc-O--R' serinol (13): To a solution of 10.0
mmol of Fmoc O--R' serine (11) in 50 mL of dry tetrahydrofuran,
kept at -20.degree. C. under nitrogen, was added 1.77 mL (12.7
mmol) of triethyl amine, followed by the slow addition of 1.57 mL
(12.0 mmol) of isobutylchloroformate. The mixture was stirred for
30 minutes, and then poured slowly over an ice-cold solution of
3.77 g (99.6 mmol) of sodium borohydride in 10 mL of water, keeping
the temperature below 5.degree. C. The reaction was stirred at
0.degree. C. for 15 minutes, and then quenched with 1N hydrochloric
acid solution. The reaction mixture was diluted with 100 mL of
ethyl acetate, and the layers separated. The organic layer was
washed with 2.times.25 mL of 1N hydrochloric acid solution,
2.times.25 mL of water, dried over magnesium sulfate and
concentrated. The compounds were purified by silica gel column
chromatography.
TABLE-US-00008 R' Analytical Data for Compounds (13) .sup.tBu
.sup.1H NMR .delta. (CDCl.sub.3): 1.14 (s, 9H, .sup.tBu), 2.90-2.95
(d, 1/2H, CH.sub.2--O), 3.63 (d, 2H, CH.sub.2--O), 3.65-3.93 (m,
3H, CH.sub.2--O), 4.20-4.35 (t, 1H, CH), 4.35-4.45 (d, 2H,
CH.sub.2), 5.50- 5.57 (d, 1H, NH), 7.26-7.8 (8H, fulvene), yield =
85%, t.sub.R = 6.42 min. Trt .sup.1H NMR .delta. (CDCl.sub.3):
3.24-3.32 (br. d, 1H, CH.sub.2--O), 3.30-3.45 (br. m, 1H,
CH.sub.2--O), 3.60- 3.987 (br. m, 3H, CH.sub.2--O, and CH--N),
4.13-4.22 (br. m, 1H, CH), 4.32-4.40 (br. d, 2H, CH.sub.2),
5.24-5.32 (br. d, 1H, NH), 7.16-7.76 (23H, fulvene, and Trt), yield
= 92%, t.sub.R = 8.39 min.
[0267] Synthesis of Fmoc-O--R' serine Weinreb amide (14): A
suspension of 20.2 mmol of Fmoc O--R' serine (11), 6.98 g (21.6
mmol) of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
tetrafluoroborate (TBTU), and 2.5 mL (22.7 mmol) of
N-methyl-morpholine in 80 mL of dry dichloromethane was stirred at
room temperature under nitrogen for 20 minutes, and then 3.02 g (31
mmol) of N,O-di-methyl-hydroxylamine hydrochloride and 3.3 mL (30
mmol) of N-methyl-morpholine were added, and the suspension stirred
at room temperature overnight. The solution formed was then
concentrated to dryness, repartitioned between 200 mL of ethyl
acetate and 100 mL of water, washed with 2.times.40 mL of 1N
hydrochloric acid solution and then 2.times.40 mL of saturated
sodium bicarbonate solution, dried over magnesium sulfate, and
concentrated. No purification was required.
TABLE-US-00009 R' Analytical Data for Compounds (14) .sup.tBu
.sup.1H NMR .delta. (CDCl.sub.3): 1.45 (s, 9H, .sup.tBu), 3.30 (s,
3H, CH.sub.3--N), 3.55-3.7 (m, 2H, CH.sub.2--O), 3.76 (s, 3H,
CH.sub.3--O), 4.19-4.26 (m, 1H, CH), 4.30-4.38 (m, 2H,
CH.sub.2--O), 4.82-4.91 (broad m, 1H, CHN), 5.68-5.75 (d, 1H, NH),
7.2-7.8 (8H, fulvene), yield = quant., t.sub.R = 6.59 min. Trt
.sup.1H NMR .delta. (CDCl.sub.3): 3.24 (s, 3H, CH.sub.3N),
3.34-3.46 (m 2H, CH.sub.2O), 3.62 (s, 3H, CH.sub.3O), 4.15-4.37
(two m, CH.sub.2, CH), 4.86-4.98 (m 1H, CHN), 5.80-5.86 (d, 1H,
NH), 7.18-7.8 (a series of m, 23H, Trt and fulvene), yield =
quant., t.sub.R = 8.0 min.
[0268] Synthesis of Fmoc-O--R' serinal (9) from ester (12): To a
solution of 3.5 mmol of (12) in 5 mL of tetrahydrofuran, kept at
-78.degree. C. under nitrogen, was added slowly 10 mL of 1N
diisobutyl aluminum hydride (DIBAL) solution, stirred for 15
minutes, and quenched by the slow addition of a saturated solution
of sodium and potassium tartrate. The reaction was allowed to warm
up to room temperature, diluted with 50 mL of ethyl acetate, and 50
mL of a saturated solution of sodium and potassium tartrate was
added. The layers were separated, and the aqueous layer
re-extracted with 1.times.50 mL of ethyl acetate. The organic
layers were combined, dried over magnesium sulfate, and
concentrated. Compounds (9) were used without further purification
in the next step.
TABLE-US-00010 R' Analytical Data for Compounds (9) .sup.tBu
.sup.1H NMR .delta. (CDCl.sub.3): 1.16 (s, 9H, .sup.tBu), 3.59-3.66
(dd, 1H, CH.sub.2O), 3.90-3.98 (dd, 1H, CH.sub.2O), 4.20-4.27 (t,
1H, CH), 4.32-4.45 (two m, 3H, CHN, and CH.sub.2O), 5.64-5.74 (br.
d, 1H, NH), 7.28-7.35 (m, 2H, fulvene), 7.36-7.44 (m, 2H, fulvene),
7.58-7.65 (d, 2H, fulvene), 7.73-7.78 (d, 2H, fulvene), 9.62 (s,
1H, CHO). Trt .sup.1H NMR .delta. (CDCl.sub.3): 3.53-3.61 (dd, 1H,
CH.sub.2O), 3.66-3.75 (dd, 1H, CH.sub.2O), 4.33-4.47 (two m, 4H,
CHN, CH, and CH.sub.2), 5.66-5.75 (d, 1H, NH), 7.20-7.81 (a series
of m, 23H, Trt, and fulvene), 9.6 (s, 1H, CHO).
[0269] Synthesis of Fmoc-O--R' serinal (9) from alcohol (13): To a
solution of 80 mmol of Fmoc-O--R' serinol (13) in 200 mL of dry
dichloromethane, kept at room temperature under nitrogen, was added
88 mmol of Dess-Martin periodinane, and the reaction was stirred
for 2.5 hours and quenched by addition of 400 mL of 10% aqueous
sodium thiosulfate solution. The layers were separated, and the
organic layer concentrated, diluted with 300 mL of ethyl ether, and
washed three times with a saturated aqueous bicarbonate solution
containing 10% sodium thiosulfate, dried over magnesium sulfate,
and concentrated.
[0270] Synthesis of Fmoc-O--R' serinal (9) from Weinreb amide (14):
To a solution of 8.8 g (20.2 mmol) of crude Fmoc-O--R' serine
Weinreb amide intermediate (14) in 60 mL of dry tetrahydrofuran,
cooled to -78.degree. C. under nitrogen, was added 30 mL of 1N
lithium aluminum hydride solution in tetrahydrofuran. The solution
was stirred for 15 minutes and then quenched by the slow addition
of 30 mL of a 1.4N solution of potassium hydrogen sulfate. After
warming up to room temperature, the solid was filtered and the
filtrate concentrated to dryness. The residue was repartitioned
between 50 mL of ethyl acetate and 25 mL of 1N hydrochloric acid
solution. The layers separated, and the organic layer was dried
over magnesium sulfate, filtered, and concentrated.
[0271] Synthesis of
(2-Fmoc-amino-3-R'--O-propylamino)-2-substituted acetic acid methyl
ester (10): compounds (10) were prepared by reductive amination
using sodium cyanoborohydride or sodium triacetoxyborohydride as
the reducing agent.
[0272] Sodium cyanoborohydride method: To a solution of 8.5 mmol of
(2) hydrochloride salt in 20 mL of methanol, kept at room
temperature under nitrogen, was added 2.3 mmol of solid potassium
hydroxide, and the mixture stirred for 25 minutes. A solution of
Fmoc-O--R' serinal (9) in 10 mL of methanol was added to the above
suspension, and the reaction mixture was stirred for 1 hour. A
solution of 8.5 mL of 1N sodium cyanoborohydride in tetrahydrofuran
was added slowly, and the reaction stirred for another 1 hour,
filtered, and concentrated. The residue was partitioned between
ethyl acetate and water, and the organic layer washed with
1.times.20 mL of saturated sodium bicarbonate, dried over sodium
sulfate, and concentrated.
[0273] Sodium triacetoxyborohydride method: A suspension of 21 mmol
of (2) hydrochloride salt, and 2.9 mL (21 mmol) of triethyl amine
in 50 mL of dry tetrahydrofuran, was stirred at room temperature
for 45 min, and then a solution of .about.20 mmol crude
Fmoc-(O--R')-serinal (9) in 30 mL of tetrahydrofuran was added,
followed by 1.7 g of 4A powdered molecular sieves, and the
suspension was stirred for an additional 2 h. 6.4 g (30 mmol) of
solid sodium triacetoxyborohydride was added, and the suspension
stirred at room temperature overnight. The suspension was diluted
with methanol, the molecular sieves filtered, and the filtrate
concentrated. The residue was partitioned between 100 mL of ethyl
acetate and 50 mL of water. The organic layer was dried over sodium
sulfate, filtered, and concentrated.
[0274] Compounds (10) were purified by silica gel column
chromatography.
TABLE-US-00011 R' R Analytical Data for Compounds (10) .sup.tBu
##STR00034## .sup.1H NMR .delta. (CDCl.sub.3): 1.17 (s, 9H,
.sup.tBu), 1.26-1.32 (d, 3H, CH.sub.3), 2.68- 2.80 (br. m, 2H,
CH.sub.2N), 3.32-3.56 (two br. m, 2H, CH.sub.2O), 3.72 (s, 3H,
CH.sub.3O), 3.66-3.82 (m, 1H, CHN), 4.18-4.28 (t, 1H, CH),
4.30-4.46 (d, 2H, CH.sub.2), 5.34-5.44 (br. d, 1H, NH), 7.25-7.44
(two m, 4H, fulvene), 7.59-7.64 (d, 2H, fulvene), 7.74-7.79 (d, 2H,
fulvene), yield = 57%, t.sub.R = 4.93 min, (M.sup.+ + 1) = 455.67.
.sup.tBu ##STR00035## .sup.1H NMR .delta. (CDCl.sub.3): 0.88-0.98
(br. t, 6H CH.sub.3), 1.21 (s 9H, .sup.tBu), 1.26- 1.34 (m, 2H,
CH.sub.2), 1.44-1.54 (m, 1H, CH), 2.58-2.86 (two m, 1H, CH.sub.2N),
3.25-3.35 (m, 1H, CH.sub.2N), 3.37-3.58 (two m, 2H, CH.sub.2O),
3.72- 3.80 (br. m, 1H, CHN), 4.14-4.31 (m, 1H, CH), 4.32-4.45 (br.
d, 2H, CH.sub.2O), 5.34-5.44 (br. d, 1H, NH), 7.30-7.84 (a series
of m, 8H, fulvene), yield = 50%, t.sub.R = 5.66 min, (M.sup.+ + 1)
= 511.67. .sup.tBu ##STR00036## .sup.1H NMR .delta. (CDCl.sub.3):
1.17 (s, 9H, .sup.tBu), 2.68-2.78 (m, 1H, CH.sub.2N), 2.82- 2.92
(m, 1H, CH.sub.2N), 3.35-3.55 (m, 4H, CH.sub.2N, and CH.sub.2O),
3.73 (s, 3H, CH.sub.3O), 3.75-3.85 (m, 1H, CHN), 4.20-4.28 (m, 1H,
CH), 4.32-4.48 (m, 2H, CH.sub.2), 5.40-5.50 (d, 1H, NH), 7.28-7.8
(a series of m, 8H, fulvene), yield = 44%, t.sub.R = 5.02 min,
(M.sup.+ + 1) = 441.50. .sup.tBu ##STR00037## .sup.1H NMR .delta.
(CDCl.sub.3): 0.84-0.92 (br. t, 3H, CH.sub.3), 1.17 (s, 9H,
.sup.tBu), 1.28- 1.35 (m, 4H, CH.sub.2), 1.48-1.84 (two m, 2H,
CH.sub.2), 2.62-2.82 (m, 2H, CH.sub.2N), 3.20-3.33 (m, 1H, CHN),
3.35-3.54 (two m, 2H, CH.sub.2O), 3.72 (s, 3H, CH.sub.3O),
3.64-3.80 (m, 1H, CHN), 4.20-4.28 (t, 1H, CH), 4.32-4.42 (m, 2H,
CH.sub.2O), 5.34-5.44 (br. d, 1H, NH), 7.25-7.79 (a series of m,
8H, fulvene), yield = 65%, t.sub.R = 5.85 min, (M.sup.+ + 1) =
441.27. Trt ##STR00038## .sup.1H NMR .delta. (CDCl.sub.3):
2.36-2.63 (br. m, 2H, CH.sub.2CO), 2.65-2.90 (br. m, 2H,
CH.sub.2N), 3.05-3.20 (br. m, 2H, CH.sub.2O), 3.50-3.64 (br. m, 1H,
CHN), 3.68 & 3.69 (two s, 3H, CH.sub.3O), 3.82-3.94 (br. m, 1H,
CHN), 4.12-4.21 (br. m, 1H, CH), 4.24-4.43 (br. m, 2H, CH.sub.2O),
4.90-4.98 (br. d, 1H, NH), 7.15-7.80 (a series of m, 23H, fulvene
and Trt), yield = 39%, t.sub.R = 8.13 min, (M.sup.+ + 1) = 926.99.
Trt ##STR00039## .sup.1H NMR .delta. (CDCl.sub.3): 1.68-1.82 (m,
1H, CH.sub.2), 1.85-1.99 (m, 1H, CH.sub.2), 2.12-2.37 (m, 2H,
CH.sub.2CO), 2.58-2.96 (a series of four m, 2H, CH.sub.2N),
3.08-3.28 (br. m, 2H, CH.sub.2O), 3.66 & 3.67 (two s, 3H,
CH.sub.3O), 3.76-3.89 (br. m, 1H, CHN), 4.15-4.24 (br. m, 1H, CH),
4.28-4.41 (br. d, 2H, CH.sub.2O), 5.10-5.22 (br. d, 1/2H, NH),
5.28-5.35 (br. d, 1/2H, NH), 7.15- 7.80 (a series of m, 23H,
fulvene, and Trt), yield = 43%, t.sub.R = 8.10 min, (M.sup.+ + 1) =
940.97. Trt ##STR00040## .sup.1H NMR .delta. (CDCl.sub.3): 1.43 (s,
6H, CH.sub.3), 1.46-1.56 (m, 4H, CH.sub.2), 2.06 (s, 3H, CH.sub.3),
2.50 (s, 3H, CH.sub.3), 2.57 (s, 3H, CH.sub.3), 2.75-2.80 (m, 1H,
CH.sub.2N), 2.91 (s, 2H, CH.sub.2), 3.12-3.32 (three br. m, 4H,
CH.sub.2N), 3.68 (s, 3H, CH.sub.3O), 4.13-4.21 (t, 1H, CH),
4.28-4.38 (d, 2H, CH.sub.2), 5.12-5.23 (br. d, 1H, NH), 5.80-6.12
(two br. m, 2H, NH), 7.18-7.80 (a series of m, 23H, fulvene, and
Trt), yield = 68%, t.sub.R = 7.52 min, (M.sup.+ + 1) = 997.91. Trt
##STR00041## .sup.1H NMR .delta. (CDCl.sub.3): 2.75-2.98 (two m,
2H, CH.sub.2N), 3.06-3.18 (m, 1H, CH.sub.2N), 3.22-3.33 (m, 1H,
CH.sub.2N), 3.57 & 3.60 (two s, 3H, CH.sub.3O), 3.80-3.92 (m,
1H, CHN), 4.00-4.08 (m, 1H, CH), 4.18-4.30 (br. d, 2H, CH.sub.2),
7.00-7.80 (a series of m, 25H, fulvene, Trt, and Imidazole), yield
= 57%, t.sub.R = 7.59 min, (M.sup.+ + 1) = 949.79. Trt ##STR00042##
.sup.1H NMR .delta. (CDCl.sub.3): 1.26 & 1.27 (two s, 9H,
.sup.tBu), 2.50-2.61 (dd, 1H, CH.sub.2-Ph), 2.76-2.86 (m, 2H,
CH.sub.2-Ph, and CH.sub.2N), 2.92-3.20 (m, 1H, CH.sub.2N),
2.92-3.20 (m, 2H, CH.sub.2O), 3.32-3.46 (m, 1H, CH.sub.2O), 3.59
(s, 3H, CH.sub.2O), 3.79-3.88 (m, 1H, CHN), 4.18-4.28 (m, 1H, CH),
4.30-4.37 (br. d, 2H, CH.sub.2O), 5.18-5.26 (br. d, 1H, NH),
6.80-6.88 (d, 2H, Ph), 6.96-7.02 (d, 2H, Ph), 7.18-7.80 (a series
of m, 23H, fulvene, and Trt), yield = 23%. Trt ##STR00043## .sup.1H
NMR .delta. (CDCl.sub.3): 1.11 (s, 9H, .sup.tBu), 2.54-2.74 (two m,
2H, CH.sub.2N), 3.02-3.58 (six m, 6H, CH.sub.2O, CH.sub.2N, and
CHN), 3.70 (s, 3H, CH.sub.3O), 3.83-3.93 (m, 1H, CHN), 4.15-4.29
(m, 1H, CH), 4.34-4.37 (d, 2H, CH.sub.2), 5.46-5.53 (br. d, 1H,
NH), 7.18-7.79 (a series of m, 23H, fulvene, and Trt), yield = 45%,
(M.sup.+ + 1) = 713.42. .sup.tTrt ##STR00044## .sup.1H NMR .delta.
(CDCl.sub.3): 0.80-0.92 (m, 7H, CH.sub.3), 1.75-1.90 (br. m, 1H,
CH), 2.6-4.36 (a series of m, 9H, CH.sub.2O, CH.sub.2N, CHN), 3.68
(s, 3H, CH.sub.3O), 5.5 (d, 0.5H, CH), 7.23-7.77 (m, 24H, fulvene
and Trt), yield = 72% (3 steps), t.sub.R = 6.86 min, (M.sup.+ + 1)
= 669.10.
[0275] Synthesis of
4-Fmoc-6-hydroxymethyl-3-substituted-piperazin-2-one (6): For the
preparation of compounds (6) three steps were required: (a) Fmoc
deprotection with concomitant cyclization, (b) Fmoc protection, and
(c) hydroxyl group deprotection.
[0276] Fmoc group removal and cyclization: A solution of 10 mmol of
cyclic compound in 30 mL of 30% diethyl amine in ethyl acetate
solution was stirred at room temperature overnight, and then
concentrated to dryness.
[0277] (a) Fmoc protection: To a biphasic solution of 10 mmol of
compound in 20 mL of tetrahydrofuran and 10 mL of water, was added
2.52 g (30 mmol) of solid sodium bicarbonate, followed by 3.36 g
(13 mmol) of Fmoc-Cl. The mixture was stirred for 3 hours, diluted
with ethyl acetate, the layers separated, and the organic layer
washed with water, dried over magnesium sulfate, and
concentrated.
[0278] (b) Hydroxyl group deprotection: For compounds containing a
.sup.tBu ether protecting group: The compounds were deprotected
with a solution of 90% trifluoroacetic acid in dichloromethane for
1-2 hours, and then concentrated to dryness. The residue was
dissolved in ethyl acetate and washed with a saturated solution of
sodium bicarbonate, dried over magnesium sulfate, and then
concentrated. For compounds containing a Trt ether protecting
group: the compounds were deprotected by adding a solution of 1-10%
trifluoroacetic acid in dichloromethane containing 2-10%
tri-isopropyl silane. The reaction was instantaneous. The solution
was then neutralized by pouring it into a saturated solution of
sodium bicarbonate. The layers were separated, dried over sodium
sulfate, and concentrated.
[0279] Compounds (6) were purified by silica gel column
chromatography.
TABLE-US-00012 R Analytical Data for Compounds (6) ##STR00045##
.sup.1H NMR .delta. (CDCl.sub.3): 1.17-1.35 (b. m, 3 H, CH.sub.3),
2.64-2.82 (t, 1 H, CH.sub.2N), 3.2-3.8 (two br. m, 3 H, CH.sub.2O,
CH.sub.2N), 4.18-4.44 (br. t, 1 H, CH), 4.64-4.90 (br. d, 2 H,
CH.sub.2O), 6.70-6.86 (br. s, 1 H, NH), 7.22-7.82 (a series of m, 8
H, fulvene), yield = 72%, t.sub.R = 4.64 min, (M.sup.+ + 1) =
367.32. ##STR00046## .sup.1H NMR .delta. (CDCl.sub.3): 0.64-1.02
(m, 6 H, CH.sub.3), 1.45-1.63 (m, 3 H, CH.sub.2, and CH), 2.65-2.84
(m, 1 H, CH.sub.2N), 2.89-3.76 (a series of br. m, 5 H, CH.sub.2O,
and CHN), 4.17-4.28 (br. m, 1 H, CH), 4.48-4.82 (three br. m,
CH.sub.2O, NH, and OH), 6.95- 7.85 (a series of br. m, 8 H,
fulvene), yield = 51%, t.sub.R = 5.43 min, (M.sup.+ + 1) = 409.08.
##STR00047## .sup.1H NMR .delta. (CDCl.sub.3): 3.17-3.78 (a series
of br. m, 5 H, CH.sub.2O, CH.sub.2N, and CHN), 421-4.27 (t, 1 H,
CH), 4.42-4.68 (br. peak, 2 H, CH.sub.2O), 6.62 (br. s, 1 H, NH),
7.28- 7.81 (a series of m, 8 H, fulvene), yield = 67%, t.sub.R =
4.50 min, (M.sup.+ + 1) = 353.45. ##STR00048## .sup.1H NMR .delta.
(CDCl.sub.3): 0.72-0.90 (br. peak, 3 H, CH.sub.3), 1.0-1.40 (br.
peak, 4 H, CH.sub.2), 1.48-1.90 (three br. peaks, 2 H, CH.sub.2),
2.68-2.80 (t, 1 H, CH.sub.2N), 3.10-3.70 (four br. peaks, 4 H,
CH.sub.2O, CHN, and CH.sub.2N), 4.15-4.25 (br. peak, 1 H, CH),
4.54-4.62 (br. d, 2 H, CH.sub.2O), 7.25-7.80 (a series of m, 8 H,
fulvene), yield = 72%, t.sub.R = 5.77 min, (M.sup.+ + 1) = 408.95.
##STR00049## .sup.1H NMR .delta. (CDCl.sub.3): 2.50-3.38 (four
overlapping br. m, 7 H, CH.sub.2--CO, CH.sub.2N, CH.sub.2O, and
CHN), 3.42-3.64 (m, 1/2 H, CHN), 3.70-3.88 (m, 1/2 H, CHN), 4.16-
4.23 (br. d, 1 H, CH), 4.48-4.68 (br. m, 2 H, CH.sub.2O), 4.94-5.05
(br. d, 1 H, NH), 6.95-7.80 (a series of m, 23 H, fulvene and Trt),
yield = 83%, t.sub.R = 7.04 min, (M.sup.+ + 1) = 652.61.
##STR00050## .sup.1H NMR .delta. (CDCl.sub.3): 1.67-1.78 (br. m, 1
H, CH.sub.2), 1.81-2.0 (br. m, 1 H, CH.sub.2), 2.10- 2.43 (br. m, 2
H, CH.sub.2--CO), 2.58-2.81 (br. m, 2 H, CH.sub.2N), 3.02-3.66 (a
series of br. m, 4 H, CH.sub.2O, and CHN), 4.17-4.23 (br. m, 1 H,
CH), 4.40-4.80 (br. m, 2 H, CH.sub.2O), 7.15-7.80 (a series of m,
23 H, fulvene, and Trt), yield = 80%, t.sub.R = 7.04 min, (M.sup.+
+ 1) = 666.66. ##STR00051## .sup.1H NMR .delta. (CDCl.sub.3): 1.43
(s, 6 H, CH.sub.3), 1.50-1.60 (br. m, 4 H, CH.sub.2), 2.10 (s, 3 H,
CH.sub.3), 2.48 (s, 3 H, CH.sub.3), 2.55 (s, 3 H, CH.sub.3), 2.92
(s, 2 H, CH.sub.2), 3.08-3.47 (two m, 3 H, CH.sub.2O, and
CH.sub.2N), 3.57-3.97 (a series of m, 3 H, CH.sub.2O, and CHN),
4.15-4.25 (br. m, 1 H, CH), 4.44-4.74 (br. m, 2 H, CH.sub.2),
7.20-7.80 (a series of br. m, 8 H, fulvene), yield = 91%, t.sub.R =
6.05 min, (M.sup.+ + 1) = 704.71. ##STR00052## .sup.1H NMR .delta.
(CDCl.sub.3): 2.14-2.56 (two m, 2 H, CH.sub.2-Im), 2.90-3.90 (a
series of m, 4 H, CH.sub.2N, and CH.sub.2O), 4.0-4.74 (a series of
m, 4 H, CHN, CH, CH.sub.2), 7.0-7.80 (a series of multiples, 25 H,
fulvene, Im, and Trt), yield = 64%, t.sub.R = 5.27 min, (M.sup.+ +
1) = 675.08. ##STR00053## .sup.1H NMR .delta. (CDCl.sub.3): 1.29
(s, 9 H, .sup.tBu) 2.47-2.74 (a series of m, 2 H, CH.sub.2Ph),
2.90-3.04 (m, 1 H, CH.sub.2Ph), 3.06-3.45 (three m, 6 H, CH.sub.2O,
and CH.sub.2N), 3.89- 4.29 (three m, 2 H, CH, and CHN), 4.32-4.42
(m, 1 H, CHN), 4.56-4.66 (m, 2 H, CH.sub.2), 6.81-7.80 (a series of
m, 12 H, fulvene, and Ph), yield = 71%, (M.sup.+ + 1) = 515.81.
##STR00054## .sup.1H NMR .delta. (CDCl.sub.3): 1.00 & 1.10 (two
s, 9 H, .sup.tBu), 3.0-3.74 (four br. m, 7 H, CH.sub.2O, CH.sub.2N,
and CHN), 3.86-4.26 (a series of m, 2 H, CHN, and CH), 4.42-4.68
(br. d, 2 H, CH.sub.2), 7.26-7.80 (a series of br. m, 8 H,
fulvene), yield = 55%, (M.sup.+ + 1) = 439.08.
[0280] Synthesis of
4-Fmoc-5-substituted-6-oxo-piperazine-2-carboxylic acid (7):
Compounds (7) were prepared as described in method A. Compounds (7)
were purified by silica gel column chromatography.
TABLE-US-00013 R Analytical Data for Compounds (7) ##STR00055##
.sup.1H NMR .delta. (CDCl.sub.3): 1.08-1.20 (br. peak, 1.5 H,
CH.sub.3), 1.30-1.38 (br. peak, 1.5 H, CH.sub.3), 2.86-3.07 (br. m,
1 H, CH.sub.2N), 3.83-3.97 (br. m, 1 H, CH.sub.2N), 4.18-4.37 (a
series of br. peaks, 2 H, CH and CHN), 4.40-4.74 (two br. peaks, 3
H, CH.sub.2O, and CHN), 7.28-7.82 (a series of m, 8 H, fulvene),
8.92-9.10 (br. s, 1 H, CO.sub.2H), yield = 51%, t.sub.R = 4.80
min,(M.sup.+ + 1) =381.57. ##STR00056## .sup.1H NMR .delta.
(CDCl.sub.3): 0.40-1.60 (a series of br. peaks, 9 H, CH, CH.sub.2,
and CH.sub.3), 2.81-3.09 (br. peak, 1 H, CH.sub.2N), 3.68-3.80 (br.
peak, 2 H, CHN), 3.96-4.32 (br. peaks, 2 H, CH, and CNH), 4.48-4.68
(br. peak, CH.sub.2O), 7.26-7.84 (a series of m, 8 H, fulvene),
yield = 50%, t.sub.R = 5.57 min, (M.sup.++ 1), = 423.15.
##STR00057## .sup.1H NMR .delta. (CDCl.sub.3): 3.77-3.99 (m, 1 H,
CHN), 3.90-4.35 (a series of m, 5 H, CH.sub.2N, CH), 4.44-4.57 (d,
2 H, CH.sub.2), 7.3-7.82 (a series of m, 8 H, fulvene), yield =
48%, t.sub.R = 4.58 min, (M.sup.+ + 1) = 367.30. ##STR00058##
.sup.1H NMR .delta. (CDCl.sub.3): 0.69-1.90 (a series of br. peaks,
CH.sub.2, and CH.sub.3), 2.85-3.05 (br. peak, 2 H, CH.sub.2N),
3.65-3.95 (two br. peaks, 1 H, CHN), 4.00-4.40 (two br. peaks,
CH.sub.2N, and CH), 4.41-4.74 (br. peak, 3 H, CH.sub.2O, and CHN),
7.20-7.80 (a series of br. m, 8 H, fulvene), yield = 70%, t.sub.R =
5.93 min, (M.sup.+ + 1) = 423.42. ##STR00059## .sup.1H NMR .delta.
(CDCl.sub.3): 2.51-3.06 (a series of m, 2 H, CH.sub.2--CO),
3.85-4.86 (a series of m, 7 H, CH.sub.2N, CHN, CH, and CH.sub.2O),
7.0-7.78 (a series of br. m, 23 H, fulvene and Trt), yield = 30%,
t.sub.R = 7.04 min, (M.sup.+ + 1) = 666.79. ##STR00060## .sup.1H
NMR .delta. (CDCl.sub.3): 1.74-2.46 (a series of br. m, 4 H,
CH.sub.2--CO, and CH.sub.2), 3.78- 4.06 (two m, 2 H, CH.sub.2N),
4.16-4.68 (a series of br. m, 5 H, CHN, CH, and CH.sub.2O),
7.14-7.82 (a series of br. m, 23 H, fulvene, and Trt), yield = 47%,
t.sub.R = 7.11 min, (M.sup.+ + 1) = 680.33. ##STR00061## .sup.1H
NMR .delta. (CDCl.sub.3): 1.08-1.60 (a series of br. peaks, 8 H,
CH.sub.2, and CH.sub.3), 2.12 (s, 3 H, CH.sub.3), 2.48 (s, 3 H,
CH.sub.3), 2.57 (s, 3 H, CH.sub.3), 2.92 (s, 2 H, CH.sub.3),
3.10-3.25 (br. m, 2 H, CH.sub.2N), 3.82-4.28 (a series of br. m, 4
H, CH.sub.2N, CHN, CH), 4.40- 4.70 (br. m, 3 H, CHN, and
CH.sub.2O), 7.20-7.80 (a series of br. m, 8 H, fulvene), yield =
42%,t.sub.R = 6.15 min, (M.sup.+ + 1) = 718.69. ##STR00062##
.sup.1H NMR .delta. (CDCl.sub.3): 1.28 & 1.34 (two s, 9 H,
.sup.tBu), 2.42-3.64 (a series of br. m, 5 H, CH.sub.2N, CHN, and
CH.sub.2Ph), 4.0-4.76 (a series of br. m, 4 H, CHN, CH, and
CH.sub.2O), 6.60-6.96 (br. m, 4 H, Ph), 7.20-7.80 (a series of br.
m, 8 H, fulvene), yield = 67%, (M.sup.+ + 1) = 529.17. ##STR00063##
.sup.1H NMR .delta. (CDCl.sub.3): 0.96- & 1.10 (two s, 9 H,
.sup.tBu), 3.04-3.18 (br. m, 0.5 H, CH.sub.2), 3.30-3.94 (four br.
m, 3.5 H, CH.sub.2N, and CH.sub.2O), 3.98-4.32 (br. m, 2 H, CH, and
CHN), 4.33-4.74 (two br. m, 3 H, CHN, CH.sub.2O), 7.28-7.80 (a
series of m, 8 H, fulvene), yield = 60%, (M.sup.+ + 1) =
453.37.
[0281] Method E:
(2-Fmoc-amino-3-hydroxy-propyl-Cbz-amino)-2-substituted acetic acid
methyl ester (15) were prepared by reductive amination of Fmoc
serinal (OR') (9) with an .alpha. amino ester (2), using either
sodium cyanoborohydride or sodium triacetoxyborohydride as the
reducing agent. The secondary amine was protected with
benzylchloroformate, and then the hydroxyl group deprotected with
trifluoroacetic acid solution. Compounds (15) were then Fmoc
deprotected. The amino ester intermediates cyclized immediately to
form 4-Cbz-3-substituted 6-hydroxymethyl-piperazin-2-ones (16).
Fmoc 3-substituted 6-hydroxymethyl-piperazin-2-ones (6) were
prepared by protecting group exchange, and then oxidized to the
desired products (7) as described in method A.
##STR00064##
[0282] Synthesis of
(2-Fmoc-amino-3-hydroxy-propyl-Cbz-amino)-2-substituted acetic acid
methyl ester (15): A suspension of 67 mmol of amino ester
hydrochloride (2), and 20.9 mmol of solid potassium hydroxide in 80
mL of methanol was stirred at room temperature for 25 minutes, and
then added to a suspension of (9) in 250 mL of methanol. The
reaction mixture was stirred for 1.5 hours, followed by the slow
addition of 70 mL of 1N sodium cyanoborohydride solution in
tetrahydrofuran. The reaction was stirred overnight, and then
concentrated. The residue was partitioned between 300 mL of
tetrahydrofuran and 50 mL of 1N hydrochloric acid solution. The
layers were separated, and the organic layer neutralized with a
solution of 239 mmol of sodium bicarbonate in 50 mL of water, and
then 66 mmol of benzyl chloroformate was added slowly, and the
reaction was stirred for 3 hours, diluted with 200 mL of ethyl
acetate, and the layers separated. The organic layer was dried over
magnesium sulfate, and concentrated. The residue was dissolved in a
solution of trifluoroacetic acid in dichloromethane, and stirred at
room temperature for 2 hours. The solution was poured over 200 mL
of saturated sodium bicarbonate solution. The layers separated, and
the organic layer was dried over magnesium sulfate, and
concentrated. Compounds (15) were purified by silica gel column
chromatography.
TABLE-US-00014 R Analytical Data for Compounds (15) ##STR00065##
.sup.1H NMR .delta. (CDCl.sub.3): 1.38-1.45 (d, 9 H, .sup.tBu),
2.68-2.78 (m, 1/2 H, CH.sub.2--CO), 3.0-3.20 (m, and s together,
3.5 H, CH.sub.2--CO, CH.sub.2--O, and CH.sub.3--O), 3.52-3.60 (m, 1
H, CH.sub.3--O), 3.96-4.40 (a series of multiples, 4 H), 4.96-5.10
(m, 2 H, CH.sub.2--O), 5.77-5.83 (m, 1/2 H, NH), 7.14-7.79, (a
series of m, 23 H, Trt and fulvene), yield = 70%, t.sub.R = 9.82
min.
[0283] Synthesis of
4-Cbz-6-hydroxymethyl-3-substituted-piperazin-2-ones (16): A
solution of 24 mmol of (15) in 100 mL of 30% diethyl amine in ethyl
acetate was stirred at room temperature overnight, and then
concentrated to dryness. The compounds were purified by silica gel
column chromatography.
TABLE-US-00015 R Analytical Data for Compounds (16) ##STR00066##
.sup.1H NMR .delta. (CDCl.sub.3): 1.36 (d, 9 H, .sup.tBu),
2.60-2.90 (m, 2 H, CH.sub.2--CO), 2.94-3.20 (br. m, 2 H, CH.sub.2N,
3.38-3.50 (br. m, 2 H, CH.sub.2--O), 3.86-4.20 (m, 1 H, CH--N),
4.74-4.84 (br, 1 H, OH), 5.10-5.15 (s, 2 H, CH.sub.2--O), 7.26-7.36
(s, 5 H, Ph), 7.87-7.95 (s, 1 H, NH), yield = 70%, t.sub.R = 4.66
min, (M.sup.+ + 1) = 379.41.
[0284] Synthesis of
4-Fmoc-6-hydroxymethyl-3-substituted-piperazin-2-ones (6): A
suspension of 15 mmol of (16), and 1.8 g of 10% palladium on carbon
in 50 mL of ethanol was hydrogenated at room temperature and
atmospheric pressure until HPLC showed that the reaction was
complete. The mixture was then filtered through celite,
concentrated, and the residue was dissolved in 35 mL of
tetrahydrofuran, and 10 mL of water, and then 62 mmol of solid
sodium bicarbonate was added, followed by 16 mmol of Fmoc-Cl, and
the mixture was stirred for 3 hours, diluted with 100 mL of ethyl
acetate and 10 mL of water. The layers were separated, and the
organic layer dried over magnesium sulfate, and concentrated.
Compounds (6) were purified by silica gel column
chromatography.
TABLE-US-00016 R Analytical Data for Compounds (6) ##STR00067##
.sup.1H NMR .delta. (CDCl.sub.3): 1.41 (s, 9 H, .sup.tBu),
2.20-2.40 (m, 1/2 H, CH.sub.2--CO, 2.64-2.96 (m, 1.5 H,
CH.sub.2--CO), 2.98-3.16 (m, 1 H, CH.sub.2O), 3.2-3.8 (a series of
br. m, 4 H, CH.sub.2O, and CH.sub.2N), 4.20-4.38 (two m, CHN, and
CH), 4.5-4.67 (br. m, 2 H, CH.sub.2O), 4.70- 4.83 (br. m, 1/2 H,
NH), 7.27-7.84 (a series of m, 8 H, fulvene), yield = 77%, t.sub.R
= 5.78 min, (M.sup.+ + 1) = 467.82.
[0285] Synthesis of
4-Fmoc-5-substituted-6-oxo-piperazine-2-carboxylic acid (7):
Compounds (7) were prepared as described in method A, and purified
by silica gel column chromatography.
TABLE-US-00017 R Analytical Data for Compounds (7) ##STR00068##
.sup.1H NMR .delta. (CDCl.sub.3): 1.4 (s, 9 H, .sup.tBu), 2.20-2.33
(br. d, 1 H, CH.sub.2--CO), 2.55-2.67 (br. d, 1 H, CH.sub.2--CO),
3.25-3.52 (br. m, 2 H, CH.sub.2N), 3.82-3.94, and 4.07-4.18 (br.
peaks, 1 H, CHN), 4.20-4.42 (m, 2 H, CHN, CH), 4.50-4.72 (m, 2 H,
CH.sub.2--O), 7.30-7.82 (8 H, fulvene), 9.20-9.35 (br. s, 1 H,
CO.sub.2H), yield = 63%, t.sub.R = 6.60 min,(M.sup.+ + 1) =
481.17.
[0286] Method F:
(2-Cbz-amino-3-benzyloxy-propylamino)-2-substituted acetic acid
methyl esters (20) were prepared by reductive amination of Cbz
serinal (OBn) (19) with an .alpha.-amino ester (2), using either
sodium cyanoborohydride or sodium triacetoxyborohydride as the
reducing agent. The Cbz O-Benzyl serinal (19) required for the
reductive amination was obtained by oxidation of Cbz serinol (OBn)
(18) with Dess-Martin periodinane. Hydrogenation of (20) followed
by cyclization gave 3-substituted 6-hydroxymethyl-piperazin-2-ones
which was then Fmoc protected to 4-Fmoc-3-substituted
6-hydroxymethyl-piperazin-2-ones (6). The final products (7) were
obtained as described in method A.
##STR00069##
[0287] Synthesis of Cbz-serinol (OBn) (18): Compound (18) was
prepared as described for compound (13). Compound (18) was obtained
in 79% yield after silica gel column chromatography purification.
.sup.1H NMR .delta. (CDCl.sub.3) 3.57-3.74 (two m, 3H, CHN, and
CH.sub.2O), 3.76-3.96 (two m, 2H, CH.sub.2O), 4.50 (s, 2H,
CH.sub.2O), 5.10 (s, 2H, CH.sub.2O), 5.40-5.50 (br. d, 1H, NH),
7.22-7.38 (m, 10H, Ph); HPLC t.sub.R=5.33 min,
(M++Na.sup.+)=337.64.
[0288] Synthesis of Cbz serinal (OBn) (19): Compound (19) was
prepared as described for compound (9). To a solution of 80 mmol of
Cbz-O-Bn serinol (18) in 200 mL of dry dichloromethane, kept at
room temperature under nitrogen, was added 88 mmol of Dess-Martin
periodinane, and the reaction stirred for 2.5 hours, and then
quenched by addition of 400 mL of 10% aqueous sodium thiosulfate
solution. The layers were separated, and the organic layer
concentrated, diluted with 300 mL of ethyl ether, and washed three
times with a saturated aqueous bicarbonate solution containing 10%
sodium thiosulfate, dried over magnesium sulfate, and concentrated.
Compound (19) was obtained in 99% crude yield, and used without
further purification. .sup.1H NMR .delta. (CDCl.sub.3) 3.69-3.78
(dd, 1H, CH.sub.2O), 3.99-4.06 (dd, 1H, CH.sub.2O), 4.37-4.46 (m,
1H, CHN), 4.47-4.52 (d, 2H, CH.sub.2O), 5.14 (s, 2H, CH.sub.2O),
5.65-5.75 (br. d, 1H, NH), 7.14-7.48 (a series of m, 9H, Ph),
7.98-8.08 (dd, 1H, Ph), 9.63 (s, 1H, CHO).
[0289] Synthesis of
(2-Cbz-amino-3-benzyloxy-propylamino)-2-substituted acetic acid
methyl esters (20): Compounds (20) were prepared as described for
compound (10), but using Cbz serinal (19) as the aldehyde.
Compounds (20) were purified by silica gel column
chromatography.
TABLE-US-00018 R Analytical Data for Compounds (20) ##STR00070##
.sup.1H NMR .delta. (CDCl.sub.3): 1.30 (s, 9 H, .sup.tBu),
2.50-2.96 (m, 3 H, CH.sub.2Ph, and CH.sub.2N), 3.28-3.54 (m, 3 H,
CH.sub.2N, and CH.sub.2O), 3.59 and 3.61 (two s, 3 H, CH.sub.3O),
3.68-3.86 (m, 1 H, CHN), 4.41-4.45 (d, 2 H, CH.sub.2O), 5.08 (s, 2
H, CH.sub.2O), 5.25-5.37 (br. t, 1 H, NH), 6.84-6.88 (d, 2 H, Ph),
6.98-7.04 (d, 2 H, Ph), 7.24-7.37 (m, 10 H, Ph), yield =
50%,(M.sup.+ + 1) = 549.35.
[0290] Synthesis of
4-Fmoc-6-hydroxymethyl-3-substituted-piperazin-2-ones (6): A
suspension of 38 mmol of (20) in 160 mL of ethanol, 38 mL of 1N
hydrochloric acid, and 20 g of 10% palladium on carbon was
hydrogenated at room temperature and atmospheric pressure until
HPLC showed that the reaction was complete. The mixture was then
filtered through celite, and concentrated to dryness. The residue
was diluted with 75 mL of tetrahydrofuran and neutralized with a
saturated sodium bicarbonate solution. 106 mmol of solid sodium
bicarbonate, and 53 mmol of Fmoc chloride were added, and the
reaction stirred at room temperature until HPLC showed the reaction
was complete, diluted with 300 mL of ethyl acetate and 300 mL of
brine. The layers were separated, and the organic layer washed
twice with brine, dried over magnesium sulfate, and concentrated.
The products (6) were purified by silica gel column
chromatography.
[0291] Synthesis of
4-Fmoc-5-substituted-6-oxo-piperazine-2-carboxylic acid (7):
Compounds (7) were prepared as described in method A.
Synthesis of 2,2-disubstituted Ketopiperazine Scaffolds Mimicking
Amino Acids Without Functionalized Side Chains (Method G)
[0292] The syntheses of
4-Fmoc-5-substituted-6-oxo-piperazine-2-methyl-2-carboxylic acid
scaffolds mimicking amino acids without functionalized side chains
was carried out using method G.
2-Boc-amino-3-methoxycarbonyl-1-substituted-methylamino-2-methyl-propioni-
c acid tert-butyl esters (23) were prepared by reductive amination
of 2-Boc-amino-2-methyl-3-oxo-propionic acid methyl ester (22) with
an .alpha.-amino ester (2), using either sodium cyanoborohydride or
sodium triacetoxyborohydride as the reducing agent. Compound (22)
required for the reductive amination was obtained by oxidation of
.alpha.-methyl-Boc serine tert-butyl ester (21) with Dess-Martin
periodinane. The Boc group of (23) was removed with 2N hydrogen
chloride in dioxane, and the amino esters cyclized to unprotected
5-substituted-6-oxo-piperazine-2-methyl-2-carboxylic acid
tert-butyl esters (24), which were protected with Fmoc chloride to
give 4-Fmoc-5-substituted-6-oxo-piperazine-2-methyl-2-carboxylic
acid tert-butyl esters, which were deprotected with trifluoroacetic
acid to give the final products (25).
##STR00071##
[0293] Synthesis of 2-Boc-amino-2-methyl-3-oxo-propionic acid
tert-butyl ester (22): Oxidation of Boc .alpha.-Methyl serine
tert-butyl ester (21) was done using Dess-Martin periodinane as
describe before gave the desired product (22) in 96% crude yield.
The compound was used without further purification in the next
step. .sup.1H NMR .delta. (CDCl.sub.3): 1.44 (s, 18H, .sup.tBu),
1.46 (s, 3H, CH.sub.3), 5.63-5.70 (br. s, 1H, NH), 9.5 (s, 1H,
CHO)
[0294] Synthesis of
2-Boc-amino-3-methoxycarbonyl-1-substituted-methylamino-2-methyl-propioni-
c acid tert-butyl ester (23): Compounds (23) were prepared using a
procedure similar to the one described for compound (10), but using
compound (22) as the aldehyde. Compounds (23) were purified by
silica gel column chromatography.
TABLE-US-00019 R Analytical Data for Compounds (23) ##STR00072##
.sup.1H NMR .delta. (CDCl.sub.3): 1.40-1.46 (two s, 21 H, CH.sub.3
and .sup.tBu), 2.60-2.72 (br. m, 1 H, CH.sub.2Ph), 2.82-3.00 (m, 3
H, CH.sub.2Ph, and CH.sub.2N), 3.32-3.43 (t, 1 H, CHN), 3.65 (s, 3
H, CH.sub.3), 5.62 (br. s, 1 H, NH), 7.13-7.32 (m, 5 H, Ph), yield
= 69%, (M.sup.+ + 1) = 436.98.
[0295] Synthesis of
2-methyl-6-oxo-5-substituted-piperazine-2-carboxylic acid (25): A
solution of 4 mmol of (23) in 8 mL of 2N hydrogen chloride in
dioxane was stirred at room temperature for 5 hours, and then
concentrated to dryness. The residue was suspended in 20 mL of
tetrahydrofuran, neutralized with 10 mmol of triethylamine, and
stirred at 60.degree. C. for 2 days. It was then concentrated to
dryness, resuspended in 20 mL of tetrahydrofuran and 10 mL of
water, solid sodium bicarbonate was added to adjust the pH to
basic, followed by 5.6 mmol of solid Fmoc chloride, and the
reaction mixture stirred overnight at room temperature, the pH
adjusted to 1 with 1N hydrochloric acid solution, diluted with 100
mL of ethyl acetate, and the layers separated. The organic layer
was washed with 2.times.100 mL of brine, dried over magnesium
sulfate and concentrated. The residue was dissolved in 10 mL of 50%
trifluoroacetic acid in dichloromethane, and the solution stirred
at room temperature for 3 hours. The solvent was concentrated, and
the products (25) purified by silica gel column chromatography.
TABLE-US-00020 R Analytical Data for Compounds (25) ##STR00073##
.sup.1H NMR .delta. (CDCl.sub.3): 1.12 (s, 3 H, CH.sub.3),
2.50-2.62 (m, 0.5 H, CH.sub.2Ph), 2.96-3.38 (three m, 1.5 H,
CH.sub.2Ph), 3.86-4.52 (a series of m, 6 H, CHN, CH, and
CH.sub.2O), 6.80-7.80 (a series of m, 13 H, fulvene and Ph), yield
= 22%, (M.sup.++ 1) = 471.47
Synthesis of 2,2-disubstituted Ketopiperazine Scaffolds Mimicking
Amino Acids with Functionalized Side Chains (Method H)
[0296] The syntheses of
4-Fmoc-5-substituted-6-oxo-piperazine-2-methyl-2-carboxylic acid
scaffolds mimicking amino acids with functionalized side chains are
performed using method H.
2-Alloc-amino-3-methoxycarbonyl-1-substituted-methylamino-2-methyl-propio-
nic acid methyl ester (30) is prepared by reductive amination of
2-Alloc-amino-2-methyl-3-oxo-propionic acid methyl ester (28) with
an .alpha.-amino allyl ester (29), using either sodium
cyanoborohydride or sodium triacetoxyborohydride as the reducing
agent, followed by protection of the secondary amine with
benzylchloroformate. Compound (28) required for the reductive
amination is obtained by oxidation of (27) with Dess-Martin
periodinane. The allyl ester and the alloc groups of analogs (30)
are removed using tetrakistriphenyl phosphine palladium (0) and the
amino acid cyclized by reaction with a peptide coupling reagent to
give 5-substituted-6-oxo-piperazine-2-methyl-2-carboxylic acid
methyl esters (31).
4-Fmoc-5-substituted-6-oxo-piperazine-2-methyl-2-carboxylic acids
(25) are obtained by saponification of the methyl ester, followed
by protecting group exchange.
##STR00074##
[0297] Synthesis of Alloc .alpha.-methyl serine methyl ester (27):
A solution of 8 mmol of Boc .alpha.-methyl serine (26), 1.0 g (12
mmol) of solid sodium bicarbonate, and 1.0 mL (16 mmol) of
iodomethane in 8 mL of dry dimethylformamide, kept under nitrogen
is stirred overnight. The reaction mixture is then poured over 50
mL of water, and extracted with 50 mL of diethyl ether, and washed
with 1.times.20 mL of water, dried over magnesium sulfate, and
concentrated. The residue is dissolved in 20 mL of 90%
trifluoroacetic acid in dichloromethane, and the solution is
stirred at room temperature for 3 hours, and then concentrated to
dryness. The residue is dissolved in 35 mL of tetrahydrofuran, and
10 ml of water, followed by addition of 30 mmol of solid sodium
bicarbonate, and the slow addition of 12 mmol of allyl
chloroformate. The mixture is stirred at room temperature for 2
hours, diluted with 50 mL of ethyl acetate, and the layers
separated. The organic layer is then washed with 1.times.10 mL of
saturated sodium bicarbonate, and 1.times.10 ml of 1N hydrochloric
acid, and 1.times.10 mL of water, dry over magnesium sulfate, and
concentrated. Compound (27) is purified by silica gel column
chromatography.
[0298] Synthesis of 2-Alloc-amino-2-methyl-3-oxo-propionic acid
methyl ester (28): Oxidation of Alloc .alpha.-methyl serine methyl
ester (27) is done using Dess-Martin periodinane as described above
to yield the desired product (28).
[0299] Synthesis of
2-Alloc-amino-3-methoxycarbonyl-1-substituted-methyl-Cbz-amino-2-methyl-p-
ropionic acid allyl ester (30): Compounds (30) are prepared using a
procedure similar to the one described for compounds (15), but
using compound (28) as the aldehyde.
[0300] Synthesis of
4-Cbz-2-methyl-6-oxo-5-substituted-piperazine-2-carboxylic acid
methyl ester (31): To solution of 10 mmol of compound (30) in 30 mL
of dichloromethane, kept at room temperature under nitrogen, is
added 2 equivalents of phenylsilane and 0.3 equivalents of
tetrakistriphenylphosphine palladium (0), and the solution stirred
for 2 hours, and then 11 mmol of TBTU, and 14 mmol of
N-methyl-morpholine are added, and the solution stirred at room
temperature for 2 hours, and then concentrated to dryness.
[0301] Synthesis of
4-Fmoc-2-methyl-6-oxo-5-substituted-piperazine-2-carboxylic acid
(25): To a solution of 10 mmol of compound (31) in 25 mL of
methanol, kept at room temperature under nitrogen, is added slowly
11 mmol of 1N sodium hydroxide solution, and the reaction is
stirred at room temperature overnight, neutralized with 21 mL of 1N
hydrochloric acid solution, 1 g of 10% palladium on carbon is
added, and the suspension hydrogenated at room temperature and
atmospheric pressure for 3 hours. The suspension is filtered
through celite and concentrated. The residue is redissolved in 25
mL of tetrahydrofuran, and 10 mL of water, followed by the addition
of 30 mmol of solid sodium bicarbonate, and 10 mmol of Fmoc
chloride, and the reaction is stirred at room temperature under
nitrogen for 2 hours. The reaction is then diluted with 50 mL of
ethyl acetate, and acidified with 1N hydrochloric acid solution.
The layers are then separated, and the organic layer is washed with
1.times.20 mL of water, dried over magnesium sulfate, and
concentrated. Compounds (25) are purified by silica gel column
chromatography.
Synthesis of (5-substituted-6-oxo-piperazin-2-yl)-acetic acid
Scaffolds (Methods I, J, K) The syntheses of
(5-substituted-6-oxo-piperazin-2-yl)-acetic acid scaffolds were
carried out by several methods.
[0302] Method I: (tert-butyl
3-protected-amino-4-(methoxycarbonyl-substituted-methylamino)-butyrates
(35) were prepared by reductive amination of tert-butyl
3-protected-amino-4-oxo-butyrate (34) with .alpha.-amino esters
(2), using either sodium cyanoborohydride or sodium
triacetoxyborohydride as the reducing agent. The tert-butyl
3-protected-amino-4-oxo-butyrate (34) required for the reductive
amination was prepared by lithium aluminum hydride (LAH) reduction
of the Weinreb amide derivatives (33). Tert-butyl
(3-protected-amino-4-(methoxycarbonyl-substituted-methylamino)-butyrate
analogs (35) were then deprotected, cyclized, and Fmoc protected to
give tert-butyl (5-substituted-6-oxo-piperazin-2-yl)-acetates (36),
which were then deprotected to give the final products (37).
##STR00075##
[0303] Synthesis of amino protected Asp-(O.sup.tBu) Weinreb amide
(33): Compounds (33) were prepared using a procedure similar to the
one described for compound (14).
TABLE-US-00021 R.sub.2 Analytical Data for Compounds (33) Cbz
.sup.1H NMR .delta. (CDCl.sub.3): 1.40 (s, 9H, .sup.tBu), 2.47-2.59
(dd, 1H, CH.sub.2CO), 3.20 (s, 3H, CH.sub.2N), 3.77 (s, 3H,
CH.sub.3O), 4.96-5.05 (br. m, 1H, CHN), 5.05-5.12 (br. d, 2H,
CH.sub.2O), 5.58-5.66 (br. d, 1H, NH), 7.30-7.36 (br. m, 5H, Ph),
yield = 90% Fmoc .sup.1H NMR .delta. (CDCl.sub.3): 1.45 (s, 9H,
.sup.tBu), 2.55-2.64 (dd, 1H, CH.sub.2CO), 2.69-2.80 (dd, 1H,
CH.sub.2O), 3.60 (s, 3H, CH.sub.3N), 3.79 (s, 3H, CH.sub.3O),
4.18-4.26 (t, 1H, CH), 4.32- 4.40 (d, 2H, CH.sub.2O), 4.98-5.19 (m,
1H, CHN), 5.70-5.76 (br. d, 1H, NH), 7.35-7.80 (a series of m, 8H,
fulvene), yield = quant.
[0304] Synthesis of tert-butyl 3-amino
protected-amino-4-oxo-butyrate (34): Compounds (34) were prepared
using a procedure similar to the one described for compound
(9).
TABLE-US-00022 R.sub.2 Analytical Data for Compounds (34) Cbz
.sup.1H NMR .delta. (CDCl.sub.3): 1.40 (s, 9H, .sup.tBu), 2.69-2.81
(dd, 1H, CH.sub.2CO), 2.89-3.01 (dd, 1H, CH.sub.2CO), 4.33-4.42 (m
1H, CHN), 5.12 (s, 2H, CH.sub.2O), 5.83-5.88 (br. d, 1H, NH),
7.31-7.39 (br. m, 5H, Ph), 9.64 (s, 1H, CHO) Fmoc .sup.1H NMR
.delta. (CDCl.sub.3): 1.45 (s, 9H, .sup.tBu), 2.58-3.02 (a series
of m, 2H, CH.sub.2CO), 4.20- 4.28 (t, 1H, CH), 4.35-4.49 (m, 3H,
CH.sub.2O, and CHN), 5.85-5.92 (br. d, 1H, NH), 7.27-7.80 (a series
of m, 8H, fulvene), 9.65 (s, 1H, CHO)
[0305] Synthesis of tert-butyl
3-Protected-amino-4-(methoxycarbonyl-substituted-methylamino)-butyrate
(35): Compounds (35) were prepared using a procedure similar to the
one described for compounds (10), but using compounds (34) as the
aldehyde.
TABLE-US-00023 R.sub.2 R Analytical Data for Compounds (35) Cbz
##STR00076## .sup.1H NMR .delta. (CDCl.sub.3): 1.40 (s, 9 H,
.sup.tBu), 2.27-3.02 (a series of m, 6 H, CH.sub.2CO, CH.sub.2Ph,
and CH.sub.2N), 3.43-3.52 (t, 1 H, CHN), 3.65 (s, 3 H, CH.sub.3O),
3.84-3.98 (m, 1 H, CHN), 5.08 (s, 2 H, CH.sub.2O), 5.33-5.44 (br.
d, 1 H, NH), 7.11-7.42 (a series of m, 10 H, Ph), yield = 60%,
t.sub.R = 4.79 min, (M.sup.+ + 1) = 471.20. Cbz ##STR00077##
.sup.1H NMR .delta. (CDCl.sub.3): 1.55 (s, 9 H, .sup.tBu),
2.42-2.68 (br. m, 2 H, CH.sub.2N), 2.74-2.92 (two dd, 2 H,
CH.sub.2O), 3.46-3.50 (d, 2 H, CH.sub.2N), 3.78 (s, 3 H,
CH.sub.3O), 4.02-4.14 (m, 1 H, CHN), 5.15 (s, 2 H, CH.sub.2O),
7.40-7.45 (m, 5 H, Ph), t.sub.R = 3.82, (M.sup.+ + 1) = 381.28 Cbz
##STR00078## .sup.1H NMR .delta. (CDCl.sub.3): 1.25-1.30 (d, 3 H,
CH.sub.3), 1.44 (s, 9 H, .sup.tBu), 2.38- 2.65 (a series of m, 2 H,
CH.sub.2CO), 2.66-2.85 (m, 2 H, CH.sub.2N), 3.60-3.70 (m, 1 H,
CHN), 3.7 (s, 3 H, CH.sub.3O), 3.9-4.1 (m, 1 H, CHN), 5.1 (s, 2 H,
CH.sub.2O), 5.4-5.6 (br. t, 1 H, NH), 7.28-7.4 (m, 5 H, Ph),
t.sub.R = 3.81 min, (M.sup.+ + 1) = 395.25. Cbz ##STR00079##
.sup.1H NMR .delta. (CDCl.sub.3): 0.84-0.91 (m, 6 H, CH.sub.3),
1.08-1.30 (m, 1 H, CH), 1.45 (s, 9 H, .sup.tBu), 1.45-1.70 (m, 2 H,
CH.sub.2), 2.39-2.60 (m, 3 H, CH.sub.2CO, CH.sub.2N), 2.74-2.86
(dd, 1 H, CH.sub.2N), 2.98-3.16 (dd, 1 H, CHN), 3.7 (s, 3 H,
CH.sub.3O), 3.92-4.08 (br. m, 1 H, CHN), 5.1 (s, 2 H, CH.sub.2O),
7.26-7.45 (m, 5 H, Ph), t.sub.R = 4.56 min,(M.sup.+ + 1) =
437.31.
[0306] Synthesis of tert-butyl
(4-Fmoc-5-substituted-6-oxo-piperazin-2-yl)-acetate (36): For
compounds containing Fmoc amino protecting group, a solution of 10
mmol of compound (35) in 30 mL of 30% diethyl amine in ethyl
acetate solution was stirred at room temperature overnight, and
then concentrated to dryness. For compounds containing Cbz amino
protecting group, a solution of 10 mmol of compound (35) in 30 mL
of ethanol was hydrogenated at room temperature and atmospheric
pressure for 2 hours, filtered through celite, and concentrated to
dryness. For Fmoc protection, the residue was dissolved in 20 mL of
tetrahydrofuran, and 10 mL of water, and 2.52 g (30 mmol) of solid
sodium bicarbonate was added, followed by the addition of 3.3 g (13
mmol) of Fmoc-Cl. The mixture was stirred for 3 hours and diluted
with ethyl acetate. The layers separated, and the organic layer was
washed with water, dried over magnesium sulfate, and concentrated.
Compounds (36) were purified by silica gel column
chromatography.
TABLE-US-00024 R Analytical Data for Compounds (36) ##STR00080##
.sup.1H NMR .delta. (CDCl.sub.3): 1.44 (s, 9 H, .sup.tBu),
1.71-2.10 (m, 2 H, CH.sub.2CO), 2.10-2.30 (br. d, 1 H, CHN),
2.62-2.82 (br. d, 1 H, CH.sub.2Ph), 2.90-3.74 (a series of br. m, 3
H, CH.sub.2N, CHN), 3.80-4.07 (br. d, 1 H, CHN), 4.10-4.50 (br. m,
3 H, CH.sub.2O, and CH), 6.74-7.80 (a series of m, 23 H, fulvene,
and Ph), yield = 75%, t.sub.R = 7.15 min, (M.sup.+ + 1) = 527.20.
##STR00081## .sup.1H NMR .delta. (CDCl.sub.3): 0.77-1.94 (a series
of m, and two s, 18 H, .sup.tBu, CH.sub.2, and CH.sub.3), 2.07-2.76
(three m, 3 H, CH.sub.2CO, and CHN), 2.86-3.80 (four m, 2 H,
CH.sub.2N), 4.16-4.27 (m, 1 H, CH), 4.30-4.43 (m, 1 H, CHN),
4.50-4.70 (br. m, 2 H, CH.sub.2O), 7.26-7.79 (a series of m, 8 H,
fulvene), yield = 40% for three steps, t.sub.R = 7.31 min,(M.sup.+
+ 1) = 493.47. ##STR00082## .sup.1H NMR .delta. (CDCl.sub.3): 1.45
(s, 9 H, .sup.tBu), 1.9-2.5 (m 2 H, CH.sub.2CO), 3.02-4.7 (a series
of m, 8 H, CH, CH.sub.2, CH.sub.2N), 7.25-7.78 (three m, 8 H,
fulvene), t.sub.R = 6.42 min, (M.sup.+ + 1) = 431.31. ##STR00083##
.sup.1H NMR .delta. (CDCl.sub.3): 1.20-1.35 (br. m, 3 H, CH.sub.3),
1.45 (s, 9 H, .sup.tBu), 2.1-2.80 (three m, 3 H, CH.sub.2CO,
CH.sub.2N), 3.1-4.1 (four m, 3 H, CH.sub.2N, CHN), 4.18-4.26 (br.
t, 1 H, CH), 4.28-4.46 (br. m, 1 H, CHN), 4.50-4.68 (br. m, 2 H,
CH.sub.2), 7.28-7.8 (three m, 8 H, fulvene), t.sub.R = 6.29 min,
(M.sup.+ + 1) = 451.24. ##STR00084## .sup.1H NMR .delta.
(CDCl.sub.3): 1.20-1.60 (br. m, and s, 15 H, CH.sub.3, .sup.tBu),
2.21-2.80 (3 br. m, 2 H, CH.sub.2CO), 3.0-3.9 (four br. m, 2 H,
CH.sub.2N), 4.18-4.26 (br. m, 2 H, CH, CHN), 4.38-4.86 (br. m, 3 H,
CH.sub.2, CHN), 7.26-7.86 (a series of m, 8 H, fulvene), t.sub.R =
6.90 min, (M.sup.+ + 1) = 493.31.
[0307] Synthesis of
(4-Fmoc-5-substituted-6-oxo-piperazin-2-yl)-acetate (37): Compounds
(36) were deprotected with 90% trifluoroacetic acid solution in
dichloromethane for 3 hours, and then concentrated to dryness.
Final products (37) were purified by silica gel column
chromatography.
TABLE-US-00025 R Analytical Data for Compounds (37) ##STR00085##
.sup.1H NMR .delta. (CDCl.sub.3): 1.82-2.13 (br. t, 1 H, CHN),
2.32-2.53 (br. d, 1 H, CH.sub.2CO). 2.63-2.81 (br. d, 1 H,
CH.sub.2CO), 2.90-3.29 (two br. m, CH.sub.2Ph), 3.38-3.59 (br. m, 1
H, CH.sub.2N), 3.66-3.85 (br. m, 1 H, CH.sub.2N), 3.95-4.24 (two
overlapping br. peaks, 2 H, CHN, CH), 4.30-4.93 (br. d, 2 H,
CH.sub.2O), 6.84-7.82 (a series of m, 13 H, fulvene, and Ph),
8.08-8.25 (br. d, 1 H,CO.sub.2H), yield = quant., t.sub.R = 5.57
min, (M.sup.+ + 1) = 471.07. ##STR00086## .sup.1H NMR .delta.
(CDCl.sub.3): 0.72-1.92 (five br. m, 9 H, CH.sub.2, and CH.sub.3) ,
2.14-2.70 (two br m, 3 H, CH.sub.2CO, and CHN), 3.26-3.62 (two br.
m, 1 H, CH.sub.2N), 3.70-3.90 (br. m, 1 H, CH.sub.2N), 4.03-4.30
(two m, 2 H, CHN, and CH), 4.42-4.82 (br. m, 2 H, CH.sub.2O),
7.28-7.82 (a series of m, 8 H, fulvene), 7.97 (s, 1 H, CO.sub.2H),
yield = 90%, t.sub.R = 5.61 min,(M.sup.+ + 1) = 437.76.
##STR00087## .sup.1H NMR .delta. (CDCl.sub.3): 2.10-2.66 (m, 2 H,
CH.sub.2CO), 3.2-3.92 (four m, 3 H, CH.sub.2N, CHN), 3.97-4.06 (m,
1 H, CH), 4.2-4.3 (m, 2 H, CH.sub.2), 4.48-4.62 (m, 2 H,
CH.sub.2N), 7.24-7.81 (a series of m, 8 H, fulvene), t.sub.R = 4.74
min, (M.sup.+ + 1) = 381.13. ##STR00088## .sup.1H NMR .delta.
(CDCl.sub.3): 1.15-1.37 (br. m, 3 H, CH.sub.3), 2.22-2.78 (three
br. m, 2 H, CH.sub.2CO), 3.0-4.10 (five br. m, 3 H, CH.sub.2N,
CHN), 4.15-4.40 (m, 1 H, CH), 4.45-4.7 (br. m, 3 H, CH.sub.2, CHN),
7.26-8.10 (a series of m, 8 H, fulvene), t.sub.R = 4.66 min,
(M.sup.+ + 1) = 395.32. ##STR00089## .sup.1H NMR .delta.
(CDCl.sub.3): 0.6-1.2 (m, 6 H, CH.sub.3), 1.22-2.8 (four m, 4 H,
CH.sub.2CO, CH.sub.2), 3.1-4.0 (five m, 3 H, CH.sub.2N, CHN),
4.18-4.32 (m, 1 H, CH), 4.41-4.84 (m, 3 H, CH.sub.2, CHN), 7.26-8.2
(a series of m, 8 H, fulvene), t.sub.R = 5.46 min, (M.sup.+ + 1) =
437.37.
[0308] Method J: Diphenylmethyl
3-Fmoc-amino-4-(methoxycarbonyl-substituted-methylamino)-butyrates
(41) were prepared by reductive amination of diphenylmethyl
3-Fmoc-amino-4-oxo-butyrate (40) with .alpha.-amino esters (2),
using either sodium cyanoborohydride or sodium
triacetoxyborohydride as the reducing agent. The diphenylmethyl
3-Fmoc-amino-4-oxo-butyrate (40) required for the reductive
amination was prepared by lithium aluminum hydride reduction of the
Weinreb amide derivative (39), which was formed from commercially
available Fmoc-aspartic acid .alpha.-allyl ester derivative (38) by
protection of the .beta.-ester under Mitsunobu conditions. The
allyl ester was removed using palladium (0) catalyst, followed by
Weinreb amide formation using TBTU as the coupling agent.
Diphenylmethyl
3-Fmoc-amino-4-(methoxycarbonyl-substituted-methylamino)-butyrate
(41) was then Fmoc deprotected, cyclized, diphenylmethyl ester
removed by hydrogenation, followed by Fmoc protection to give the
final product (4-Fmoc-5-substituted-6-oxo-piperazin-2-yl)-acetic
acid (37).
##STR00090##
[0309] Synthesis of Fmoc-Asp-(OCHPh.sub.2) Weinreb amide (39): To a
solution of 5.1 g (13.0 mmol) of Fmoc-aspartic acid .alpha.-allyl
ester (38) in 30 mL of dry tetrahydrofuran, containing 3.4 g (13
mmol) of triphenylphosphine, and 2.41 g (13.1 mmol) of
diphenylmethanol, kept at 0.degree. C. under nitrogen, was added
slowly 2.6 mL (13.4 mmol) of diisopropyl azodicarboxylate. The ice
bath was removed, and the reaction stirred at room temperature
overnight, concentrated to dryness, and then purified by silica gel
column chromatography. .sup.1H NMR .delta. (CDCl.sub.3) 2.96-3.06
(dd, 1H, CH.sub.2CO), 3.15-3.26 (dd, 1H, CH.sub.2CO), 4.18-4.76 (a
series of m, 3H, CH, CH.sub.2), 5.14-5.32 (m, 1H, CHN), 5.76-5.86
(m, 1H, CHO), 7.20-7.80 (a series of m, 18H, fulvene, and Ph); HPLC
t.sub.R=7.68 min, (M.sup.++Na.sup.+)=583.90.
[0310] The product (9.8 mmol) was then dissolved in 40 mL of a
dichloromethane:acetic acid:N-methyl morpholine solution at 37:2:1,
containing 1.5 g (1.3 mmol) of tetrakis triphenylphosphine
palladium (0), and the solution stirred at room temperature
overnight, concentrated to dryness, and partitioned between 100 mL
of ethyl acetate and 30 mL of water. The layers were separated, and
the organic layer washed with 1.times.50 mL of water, dried over
sodium sulfate, and concentrated. The residue was suspended in 20
mL of dry dichloromethane, and 1.65 mL (15 mmol) of N-methyl
morpholine, and 4.07 g (12.7 mmol) of TBTU were added, and the
suspension stirred at room temperature for 20 minutes, followed by
the addition of 1.65 mL (15 mmol) of N-methyl morpholine, and 1.52
g (15.6 mmol) of N,O-dimethyl hydroxylamine hydrochloride salt. The
suspension was stirred at room temperature for 2 hours,
concentrated, partitioned between 100 mL of ethyl acetate and 50 mL
of water. The organic layer was washed with 1.times.30 mL of water,
1.times.30 mL of saturated sodium bicarbonate solution, and
1.times.30 mL of 1N hydrochloric acid solution, dried over sodium
sulfate, and concentrated. The product was purified by silica gel
column chromatography. .sup.1H NMR .delta. (CDCl.sub.3) 2.76-2.88
(dd, 1H, CH.sub.2CO), 2.89-3.00 (dd, 1H, CH.sub.2CO), 3.16 (s, 3H,
CH.sub.3N), 3.70 (s, 3H, CH.sub.3O), 4.14-4.22 (dd, 1H, CH),
4.28-4.40 (t, 2H, CH.sub.2), 5.07-5.16 (dd, 1H, CHN), 5.69-5.76 (d,
1H, CHO), 7.24-7.8 (a series of m, 18H, fulvene, and Ph); HPLC
t.sub.R=7.08, (M++Na.sup.+)=587.03.
[0311] Synthesis of Diphenylmethyl 3-Fmoc-amino-4-oxo-butyrate
(40): Compound (40) is prepared using a procedure similar to the
one described for compound (9).
[0312] Synthesis of Diphenylmethyl
3-Fmoc-amino-4-(methoxycarbonyl-substituted-methylamino)-butyrate
(41): Compounds (41) were prepared using a procedure similar to the
one described for compound (10), but using compound (40) as the
aldehyde.
TABLE-US-00026 R Analytical Data for Compounds (41) ##STR00091##
.sup.1H NMR .delta. (CDCl.sub.3) 1.2-1.7 (m, 4 H, CH.sub.2), 1.42
(s, 3 H, CH.sub.3Ph), 1.60 (s, 6 H, CH.sub.3- Ph), 2.07 (s, 2 H,
CH.sub.2), 2.52 (s, 3 H, CH.sub.3-Ph), 2.58 (s, 3 H, CH.sub.3-Ph),
2.08-2.80 (a, series of m, 2 H, CH.sub.2CO), 3.0-3.2 (m, 2 H,
CH.sub.2N), 3.64 (s, 3 H, CH.sub.3O), 3.96-4.10 (m, 1 H, CHN),
4.20-4.28 (m, 1 H, CH), 4.28-4.40 (br. m, 2 H, CH.sub.2), 5.82-6.18
(m, 1 H, CHO), 7.24-7.80 (a series of m, 18 H, fulvene, and Ph),
HPLC t.sub.R = 6.53, (M.sup.+ + 1) = 930.56.
[0313] Synthesis of
(4-Fmoc-5-substituted-6-oxo-piperazin-2-yl)-acetic acid (37): A
solution of 10 mmol of compound (41) in 30 mL of 30% diethylamine
in ethyl acetate was stirred at room temperature for 3 hours. The
solution was then concentrated to dryness, redissolved in
2.times.30 mL of ethyl acetate, and reconcentrated. The residue
dissolved in 50 mL of ethanol, and 20 mL of 1N hydrochloric acid
solution, and hydrogenated at room temperature and atmospheric
pressure overnight, filtered through celite, and concentrated to
dryness. The residue was dissolved in 20 mL of tetrahydrofuran, and
10 mL of water, and 2.52 g (30 mmol) of solid sodium bicarbonate
was added, followed by the addition of 3.3 g (13 mmol) of Fmoc-Cl.
The mixture was stirred for 3 hours, diluted with 100 mL of ethyl
acetate, the layers separated, and the organic layer washed with
2.times.50 mL of water, dried over magnesium sulfate, and
concentrated. The product was purified by silica gel column
chromatography.
TABLE-US-00027 R Analytical Data for Compounds (37) ##STR00092##
.sup.1H NMR .delta. (CDCl.sub.3) 1.2-1.6 (m, and s, 7 H, CH.sub.2,
CH.sub.3Ph), 2.10 (s, 2 H, CH.sub.2), 2.46 (s, 3 H, CH.sub.3-Ph),
2.56 (s, 3 H, CH.sub.3-Ph), 2.46-2.63 (br. m, 2 H, CH.sub.2CO),
3.0-3.95 (3 b. m, 5 H, CH.sub.2N, CHN), 4.10-4.30 (br. m, 1 H, CH),
4.40-4.80 (br. m, 3 H, CHN, CH.sub.2), 7.22-7.80 (a series of m, 8
H, fulvene), HPLC t.sub.R = 5.73, (M.sup.+ + 1) 732.24.
[0314] Method K: The syntheses of
(5-substituted-6-oxo-piperazin-2-yl)-acetic acid scaffolds are done
starting from commercially available Fmoc-Aspartic acid .alpha.
tert-butyl ester (42). Fmoc-aspartic acid .alpha. tert-butyl ester
is reduced to Fmoc-Homoserine .alpha. tert-butyl ester with sodium
borohydride via the mixed anhydride, followed by protection of the
alcohol with benzyl bromide to give Fmoc-Homoserine benzyl ether
.alpha. tert-butyl ester (43). The tert-butyl ester is then removed
with trifluoroacetic acid, and the acid is reduced to the alcohol
with sodium borohydride via the mixed anhydride to give
2-Fmoc-amino-4-benzyloxy-1-butanol (44). Alcohol (44) is then
converted to 2-Fmoc-amino-4-benzyloxybutanal (45) using Dess-Martin
periodinane as described previously. Reductive amination of
2-Fmoc-amino-4-benzyloxybutanal (45) and .alpha.-amino ester (2)
gives the (2-Fmoc-amino-4-benzyloxy-butylamino)-2-substituted
acetic acid methyl ester (46). Fmoc deprotection with diethyl amine
gives the free primary amine which cyclizes to
6-benzyloxyethyl-3-substituted-piperazin-2-one spontaneously. The
benzyl ether is removed by hydrogenation, and the secondary amine
is protected as its Fmoc derivative to give
4-Fmoc-6-hydroxymethyl-3-substituted-piperazin-2-ones (47).
Finally, the primary alcohol is oxidized to the acid to give the
final products (48) as described in method A.
##STR00093##
[0315] Synthesis of Fmoc-Homoserine (OBn) .alpha. tert-butyl ester
(43): To a solution of 10.0 mmol of Fmoc Asp-O.sup.tBu (42) in 50
mL of dry tetrahydrofuran, kept at -20.degree. C. under nitrogen,
is added 1.77 mL (12.7 mmol) of triethyl amine, followed by the
slow addition of 1.57 mL (12.0 mmol) of isobutylchloroformate. The
mixture is stirred for 30 minutes, and then poured slowly over an
ice-cold solution of 3.77 g (99.6 m mol) of sodium borohydride in
10 mL of water, keeping the temperature below 5.degree. C. The
reaction is stirred at 0.degree. C. for 15 minutes, and then
quenched with 1N hydrochloric acid solution. The reaction mixture
is diluted with 100 mL of ethyl acetate, and the layers separated.
The organic layer was washed with 2.times.25 mL of 1N hydrochloric
acid solution, 2.times.25 mL of water, dried over magnesium sulfate
and concentrated, and purified by silica gel column chromatography.
Purified compound is then dissolved in 30 mL of tetrahydrofuran,
and 12 mmol of 60% sodium hydride dispersion in mineral oil is
added, followed by 0.2 mmol of tetrabutylammonium iodide and 12
mmol of benzyl bromide, and the mixture is stirred overnight,
quenched with 50 mL of saturated aqueous sodium bicarbonate, and
extracted with 100 mL of ethyl acetate. The compound is then
purified by silica gel column chromatography.
[0316] Synthesis of 2-Fmoc-amino-4-benzyloxy-1-butanol (44):
Deprotection of the tert-butyl ester using 90% trifluoroacetic acid
is done as described for compound (37) in method I, followed by
reduction of the acid to the alcohol with sodium borohydride via
the mixed anhydride intermediate as described for compound
(13).
[0317] Synthesis of 2-Fmoc-amino-4-benzyloxy-butanal (45):
2-Fmoc-amino-4-benzyloxy-1-butanol (44) is oxidized to the aldehyde
using Dess-Martin periodinane as described for the synthesis of
(9).
[0318] Synthesis of
(2-Fmoc-amino-4-benzyloxy-butylamino)-2-substituted acetic acid
methyl ester (46): reductive amination of
2-Fmoc-amino-4-benzyloxy-butanal (45) with an .alpha.-amino ester
(2) using either sodium cyanoborohydride or sodium
triacetoxyborohydride as the reducing agent is done as described
for the synthesis of (10).
[0319] Synthesis of
4-Fmoc-6-hydroxymethyl-3-substituted-piperazin-2-ones (47): Fmoc
deprotection of (2-Fmoc-amino-4-benzyloxy-butylamino)-2-substituted
acetic acid methyl ester (46) with concomitant cyclization,
followed by de-benzylation and Fmoc reprotection is done as
described for compound (37) in method J.
[0320] Synthesis of
4-Fmoc-5-substituted-6-oxo-piperazin-2-yl-acetic acid (37):
Oxidation of 4-Fmoc-6-hydroxymethyl-3-substituted-piperazin-2-ones
(47) to the acid is done as described in method A. The choice of
the oxidizing agent used is based on the nature of the group in the
5-position.
Synthesis of 2-Substituted 3-Oxo-[1,4]-diazepane-5-carboxylic acid
Scaffolds (Methods L, M, N)
[0321] The synthesis of 2-substituted
3-oxo-[1,4]-diazepane-5-carboxylic acid scaffolds is done using
several methods.
[0322] Method L: tert-butyl
2-Cbz-amino-4-(benzyloxycarbonyl-substituted-methyl-Boc
amino)-butyrates (52) are prepared by reductive amination of
tert-butyl Cbz-2-amino-4-oxo-butyrate (50) with amino ester (51),
using either sodium cyanoborohydride or sodium
triacetoxyborohydride as the reducing agent, followed by Boc
protection of the secondary amine. The tert-butyl
Cbz-2-amino-4-oxo-butyrate (50) required for the reductive
amination is prepared by lithium aluminum hydride reduction of the
Weinreb amide derivative (49). The diazepane ring is formed by
protecting group removal, followed by cyclization with a peptide
forming reagent to give (53). Finally, 4-Fmoc-2-substituted
3-oxo-[1,4]-diazepane-5-carboxylic acids (54) are formed by
protecting group exchange.
##STR00094##
[0323] Synthesis of Cbz-Asp-(Weinreb amide)-O.sup.tBu (49):
Compound (49) is prepared using a procedure similar to the one
described for compound (14).
[0324] Synthesis of tert-butyl 3-Cbz-amino-4-oxo-butyrate (50):
Compound (50) is prepared using a procedure similar to the one
described for compound (9).
[0325] Synthesis of tert-butyl
2-Cbz-amino-4-(benzyloxycarbonyl-substituted-methyamino)-butyrate
(52): The reductive amination is done with procedure similar to the
one described for compound (10). The secondary amine is protected
by reaction of the crude mixture with 2 equivalents of Boc
dicarbonate in tetrahydrofuran.
[0326] Synthesis of tert-butyl 1-Boc
2-substituted-3-oxo-[1,4]-diazepane-5-carboxylate (53): A solution
of 10 mmol of compound (52) in 30 mL of ethanol is hydrogenated at
room temperature and atmospheric pressure for 2 hours, filter
through celite, and concentrated to dryness. The residue is
dissolved in 100 mL of dichloromethane and 1.2 equivalents of TBTU,
and 2.6 equivalents of N-methyl-morpholine are added. The solution
is stirred at room temperature overnight, and then concentrated.
The residue is partitioned between 50 mL of ethyl acetate and 25 mL
of 1N hydrochloric acid solution, washed with 1.times.20 mL of a
saturated sodium bicarbonate solution, dried over magnesium
sulfate, and concentrated.
[0327] Synthesis of 1-Fmoc
2-substituted-3-oxo-[1,4]-diazepane-5-carboxylic acid (54): A
solution of 10 mmol of compound (53) in 10 mL of 90%
trifluoroacetic acid in dichloromethane is stirred at room
temperature for 2 hours, and then the solution is concentrated to
dryness. The residue is dissolved in 20 mL of tetrahydrofuran and
10 mL of water, and 2.52 g (30 mmol) of solid sodium bicarbonate is
added, followed by the addition of 3.36 g (13 mmol) of Fmoc-Cl. The
mixture is stirred for 3 hours, and then diluted with ethyl
acetate. The layers are separated, and the organic layer washed
with 2.times.50 mL of water, dried over magnesium sulfate, and
concentrated.
[0328] Method M: the reduced dipeptide analogs (60) are prepared by
reductive amination of diphenylmethyl Alloc-2-amino-4-oxo-butyrate
(59) with amino ester (29), using either sodium cyanoborohydride or
sodium triacetoxyborohydride as the reducing agent, followed by Cbz
protection of the secondary amine. Diphenylmethyl
Alloc-2-amino-4-oxo-butyrate (59) required for the reductive
amination is prepared by lithium aluminum hydride reduction of the
Weinreb amide derivative (58), which is prepared by protecting
group exchange of Weinreb amide derivative (57). The diazepane ring
is then formed by allyl and alloc group removal, followed by ring
closure in the presence of a peptide forming reagent. 2-substituted
3-oxo-[1,4]-diazepane-5-carboxylic acid scaffolds (54) are formed
by protecting group exchange.
##STR00095##
[0329] Synthesis of Fmoc-Asp-(Weinreb amide)-OCHPh.sub.2 (57):
Compound (57) is prepared using a procedure similar to the one
described for compound (39).
[0330] Synthesis of Alloc-Asp-(Weinreb amide)-OCHPh.sub.2 (58): A
solution of 10 mmol of compound (56) in 20 mL of 30% diethylamine
in ethyl acetate is stirred for 2 hours, and concentrated to
dryness. The residue is dissolved in 20 mL of tetrahydrofuran and
10 mL of water, and 2.52 g (30 mmol) of solid sodium bicarbonate is
added, followed by the addition of 13 mmol of Alloc-Cl. The mixture
is stirred for 3 hours, and then diluted with ethyl acetate. The
layers are separated, and the organic layer washed with water,
dried over magnesium sulfate, and concentrated. Compound (58) is
purified by silica gel column chromatography.
[0331] Synthesis of diphenylmethyl 3-Alloc-amino-4-oxo-butyrate
(59): Compound (59) is prepared using a procedure similar to the
one described for compound (9).
[0332] Synthesis of diphenyl methyl
2-Alloc-amino-4-(allyloxycarbonyl-substituted-methyamino)-butyrate
(60): compound 60 is prepared by reductive amination using a
procedure similar to the one described for compounds (15), but
using compound (59) as the aldehyde. The product is purified by
silica gel column chromatography.
[0333] Synthesis of diphenylmethyl 1-Cbz
2-substituted-3-oxo-[1,4]-diazepane-5-carboxylate (61): To a
solution of 10 mmol of compound (60) in 30 mL of dichloromethane,
kept at room temperature under nitrogen, is added 2 equivalents of
phenylsilane and 0.3 equivalents of tetrakistriphenylphosphine
palladium (0), and the solution stirred for 2 hours, and then 1.2
equivalents of TBTU and 1.3 equivalents of N-methyl-morpholine are
added. The solution is stirred at room temperature overnight and
concentrated. The residue is partitioned between 50 mL of ethyl
acetate and 25 mL of 1N hydrochloric acid solution, washed with
1.times.20 mL of a saturated sodium bicarbonate solution, dried
over magnesium sulfate, and concentrated.
[0334] Synthesis of 1-Fmoc
2-substituted-3-oxo-[1,4]-diazepane-5-carboxylic acid (54): A
solution of 10 mmol of compound (61) in 30 mL of ethanol is
hydrogenated at room temperature for 2 hours, filtered through
celite, and then the solution is concentrated to dryness. The
residue is dissolved in 20 mL of tetrahydrofuran, and 10 mL of
water, and 2.52 g (30 mmol) of solid sodium bicarbonate is added,
followed by the addition of 3.36 g (13 mmol) of Fmoc-Cl. The
mixture is stirred for 3 hours, and then diluted with ethyl
acetate. The layers are separated, and the organic layer washed
with water, dried over magnesium sulfate, and concentrated.
[0335] Method N: Fmoc-Aspartic acid .beta. tert-butyl ester is
reduced to Fmoc-Aspartanol .beta. tert-butyl ester (63) with sodium
borohydride via the mixed anhydride, followed by protection of the
alcohol with allyl bromide to give Fmoc-Aspartanol allyl ether
.beta. tert-butyl ester (64). The tert-butyl ester is then removed
with trifluoroacetic acid, and the acid reduced to the alcohol with
sodium borohydride via the mixed anhydride to give
3-Fmoc-amino-4-allyloxy-1-butanol (65). Alcohol (65) is then
converted to 3-Fmoc-amino-4-allyloxybutanal (66) using Dess-Martin
periodinane as described previously. Reductive amination of
3-Fmoc-amino-4-allyloxybutanal (66) and .alpha. amino ester (51),
followed by alloc protection on the secondary amine, gives the
(3-Fmoc-amino-4-allyloxy-butyl-alloc-amino)-2-substituted acetic
acid benzyl esters (67). Alloc
7-allyloxymethyl-3-substituted-[1,4]-diazepan-2-ones (68) are
formed by saponification of the benzyl ester, followed by Fmoc
deprotection with diethyl amine to give the free primary amine
which is cyclized using a peptide forming reagent such as TBTU. The
final products (54) are formed by protecting group exchange: the
allyl ether and the alloc are removed by palladium (0), and the
secondary amine is protected as its Fmoc derivative to give
4-Fmoc-7-benzyloxymethyl-3-substituted-[1,4]-diazepan-2-ones,
followed by primary alcohol oxidation to the acid to give the final
products (54). The choice of the oxidizing agent used is based on
the nature of the group in the 2-position.
##STR00096##
[0336] Synthesis of Fmoc-Aspartanol .beta. tert-butyl ester (63):
Compound (63) is prepared as described for the synthesis of
compound (13), using Fmoc-Aspartic acid .beta. tert-butyl ester
(62) as the starting material.
[0337] Synthesis of 3-Fmoc-amino-4-allyloxy-butyric acid tert-butyl
ester (64): To a solution of 10 mmol of (63) in 30 mL of
tetrahydrofuran, kept at room temperature under nitrogen, is added
12 mmol of 60% sodium hydride dispersion in mineral oil, 2 mmol of
tetrabutylammonium iodide, and 13 mmol allyl bromide, and the
mixture is stirred overnight, quenched with 10 mL of saturated
aqueous sodium bicarbonate, and extracted with 50 mL of ethyl
acetate.
[0338] Synthesis of 3-Fmoc-amino-4-allyloxy-1-butanol (65):
Compound (65) is prepared as described for the synthesis of
compound (44).
[0339] Synthesis of 3-Fmoc-amino-4-allyloxy-butanal (66):
3-Fmoc-amino-4-allyloxy-1-butanol (65) is oxidized to the aldehyde
using Dess-Martin periodinane as described for the synthesis of
(9).
[0340] Synthesis of
(3-Fmoc-amino-4-allyloxy-butyl-alloc-amino)-2-substituted acetic
acid methyl ester (67): reductive amination of
3-Fmoc-amino-4-benzyloxy-butanal (66) with an .alpha.-amino ester
(51) using either sodium cyanoborohydride or sodium
triacetoxyborohydride as the reducing agent as described for
compound (10), followed by protection of the secondary amine as the
alloc derivative, is done as described for compound (15), but using
allyl chloroformate instead of benzyl chloroformate.
[0341] Synthesis of
4-Alloc-7-allyloxymethyl-3-substituted-[1,4]-diazepan-2-ones (68):
A solution of 10 mmol of
(3-Fmoc-amino-4-allyloxy-butyl-alloc-amino)-2-substituted acetic
acid methyl ester (67), 20 mmol of potassium carbonate in 20 mL of
methanol, and 10 mL of water is stirred at room temperature for 3
hours, neutralized with 21 mL of a 1N hydrochloric acid solution,
and then concentrated to dryness. The residue is dissolved in 20 mL
of 30% diethyl amine in ethyl acetate and stirred at 3 hours, and
then concentrated to dryness. The residue is dissolved in 100 mL of
dichloromethane, and 12 mmol of TBTU and 24 mmol of
N-methylmorpholine are added, and the solution stirred at room
temperature overnight, and then concentrated to dryness. The
residue is partitioned between 30 mL of ethyl acetate and 30 mL of
1N hydrochloric acid solution, and then the layers separated. The
organic layer is washed with 30 mL of a saturated sodium
bicarbonate solution, dried over magnesium sulfate, and purified by
silica gel column chromatography.
[0342] Synthesis of
4-Fmoc-2-substituted-3-oxo-[1,4]-diazepane-5-carboxylic acid (54):
To solution of 10 mmol of compound (68) in 30 mL of
dichloromethane, kept at room temperature under nitrogen, is added
2 equivalents of phenylsilane and 0.3 equivalents of
tetrakistriphenylphosphine palladium (0), and the solution then
stirred for 2 hours, and concentrated to dryness. The secondary
amine is dissolved in 20 mL of tetrahydrofuran, and 10 mL of water,
followed by the addition of 2.52 g (30 mmol) of solid sodium
bicarbonate, and 1.2 equivalents of Fmoc-Cl and the biphasic
solution is stirred at room temperature for 2 hours, diluted with
30 mL of ethyl acetate, and the layers separated. Oxidation of
4-Fmoc-7-hydroxymethyl-3-substituted-[1,4]-diazepan-2-ones to the
final product (54) is done as described in method A. The choice of
the oxidizing agent used is based on the nature of the group in the
2-position, as in Method A for the conversion of (6) to (7).
Synthesis of 6-substituted-5-oxo-piperazine-2-carboxylic acid
Scaffolds (Method O)
[0343] The syntheses of 6-substituted-5-oxo-piperazine-2-carboxylic
acid scaffolds containing non-functionalized side chains in the
6-position are done as outlined in Method O, starting from
commercially available 3-Fmoc-amino-1,2-propan-diol 1-chloro-trityl
resin (69) which is oxidized to the ketone (70) using Dess-Martin
periodinane. Reductive amination of ketone (70) with an .alpha.
amino ester (2) gives resin bound
(1-aminomethyl-2-chloro-trityloxy-ethylamino)-2-substituted acetic
acid methyl ester (71), which is cyclized to
5-chlorotrityloxymethyl-3-substituted-piperazin-2-one (72) after
deprotection of the amine. Reprotection of the secondary amine,
followed by cleavage from the resin, gives
Fmoc-5-hydroxymethyl-3-substituted-piperazin-2-one (73) which is
oxidized to 6-substituted-5-oxo-piperazine-2-carboxylic acid (74)
using either of the procedures described in method A.
##STR00097##
[0344] Synthesis of 1-amino-3-chlortrityloxy-propan-2-one (70): the
oxidation of resin bound alcohol (69) is done by sulfur trioxide
oxidation, NMO/TPAP (N-methylmorpholine-N-oxide/tetrapropyl
ammonium perrthenate) oxidation, or PDC oxidation. For sulfur
trioxide oxidation, a procedure similar to the one described in
Parikh, J. R. and Doering, W. V., J. Am. Chem. Soc. 89:5505-5507
(1967) is used. For NMO/TPAP oxidation, to 0.3 mmol of resin-bound
alcohol is added a solution of 3 mmol of N-methylmorpholine N-oxide
in 10 mL of dry dimethylformamide, and then 0.06 mmol of
tetrapropylammonium perruthenate (TPAP) is added to the resin
suspension. The reaction is shaken for 80 minutes. The solvent is
drained, the resin washed with tetrahydrofuran and dichloromethane,
and then dried under vacuum. For PDC oxidation, a suspension of
resin bound alcohol in 0.2 M pyridinium dichromate in
dimethylformamide is shaken at 37.degree. C. for 4 hours, the
solvent is drained, and the resin washed with dimethylformamide,
tetrahydrofuran, and dichloromethane.
[0345] Synthesis of
(1-aminomethyl-2-chloro-trityloxy-ethylamino)-2-substituted acetic
acid methyl ester (71): the reductive amination of resin bound
ketone (70) with amino ester is done by one of two different
methods. In one method, a solution of 2.6 mmol of .alpha. amino
ester (2) in 20 mL of 1% acetic acid in dimethylformamide is added
2.6 mmol of sodium triacetoxyborohydride, followed by the immediate
addition of 0.5 mmol of ketone-derivatized resin (70), and the
mixture is shaken for 60 minutes, rinsed with methanol, 10%
di-isopropyl ethyl amine, dimethylformamide, and methanol. In a
second method, a suspension of 0.05 mmol of ketone-derivatized
resin (70) and 2.0 M .alpha. amino ester hydrochloride (2) in
methanol, containing 0.05 M sodium cyanoborohydride is shaken at
room temperature for 5 hours, drained, and washed.
[0346] Synthesis of
5-chlorotrityloxymethyl-3-substituted-piperazin-2-one (72): A
suspension of 0.05 mmol of resin in 10 mL of 20% piperidine in
dimethylformamide is shaken at room temperature for 2 hours.
[0347] Synthesis of
Fmoc-5-hydroxymethyl-3-substituted-piperazin-2-one (73): A
suspension of 0.05 mmol of (72) in 10 mL of dichloromethane,
containing 0.25 mmol of Fmoc-Cl and 0.25 mmol of triethyl amine is
stirred at room temperature for 6 hours, drained, and washed with
dichloromethane. The resin is resuspended in 10 mL of 95%
trifluoroacetic acid in dichloromethane, and the suspension shaken
for 2 hours, and filtered, and the filtrate is concentrated.
[0348] Synthesis of
Fmoc-6-substituted-5-oxo-piperazine-2-carboxylic acid (74):
Oxidation of (73) to the desired product is done by any of the
procedures described for method A.
Synthesis of .alpha., .alpha.-Disubstituted Amino Acids (Methods P
and Q)
[0349] In certain of the constructs of the invention, it is
possible and contemplated to employ a disubstituted amino acid
residue, such as an .alpha., .alpha.-disubstituted amino acid where
the substituents are either the same or different. In one aspect,
an .alpha., .alpha.-disubstituted amino acid is employed in either
the Aaa.sup.1 or Aaa.sup.8 position, wherein at least one of the
side chains of the .alpha., .alpha.-disubstituted amino acid is a
side chain of Nle, Ala, Leu, Ile, Val, Nva, Met(O) or Met(O.sub.2).
The following synthetic Methods P and Q describe making .alpha.,
.alpha.-di-n-butylglycine (2-Amino-2-butyl-hexanoic acid), wherein
each of the side chains are --(CH.sub.2).sub.3--CH.sub.3, and thus
each is the same as the side chain of Nle. However, it is to be
understood that similar methods and schemes may be employed in the
making of other .alpha., .alpha.-disubstituted amino acids, where
the substituents are either the same or different. Additionally,
any method of making an .alpha., .alpha.-disubstituted amino acid
may be employed in the practice of this invention, and the practice
of this invention is not limited to the methods of the following
synthetic schemes. Thus any method known in the art for the
synthesis of .alpha., .alpha.-disubstituted amino acids may be
employed in the practice of this invention. The following teach
alternative methods for the making of .alpha.,
.alpha.-disubstituted amino acids: Clark J. S. and Middleton M. D.:
Synthesis of novel alpha-substituted and alpha,alpha-disubstituted
amino acids by rearrangement of ammonium ylides generated from
metal carbenoids. Org. Lett. 4(5):765-8 (2002); Guino M., Hii K.
K.: Wang-aldehyde resin as a recyclable support for the synthesis
of alpha,alpha-disubstituted amino acid derivatives. Org. Biomol.
Chem. 3(17):3188-93 (2005); and Kotha S., Behera M.: Synthesis and
modification of dibenzylglycine derivatives via the Suzuki-Miyaura
cross-coupling reaction. J. Pept. Res. 64(2):72-85 (2004).
##STR00098##
[0350] Synthesis of Benzoyl di-n-butylglycine (80): To a solution
of 10 mmol benzoyl glycine (75) in 20 mL of dichloromethane, kept
at 0.degree. C. under nitrogen, is added slowly 12 mmol of
N,N'-dicyclohexylcarbodiimide (DCC), and the reaction stirred for 2
hours to yield compound (76). The solid is filtered off, and the
filtrate concentrated. The residue is dissolved in 15 mL of
tetrahydrofuran, cooled to 0.degree. C., and then 24 mmol of sodium
hydride is added, followed by 30 mmol of n-butyl bromide. The
suspension is stirred at 0.degree. C. for 2 hours and then allowed
to warm to room temperature, and the solution concentrated to
dryness to yield compound (77). Alternatively, compound (77) can
also be prepared from benzoyl norleucine (78) in a similar manner
except that 12 mmol of sodium hydride and 15 mmol of n-butyl
bromide are used. Compound (77) is dissolved in methanol, 50 mL of
1N hydrochloric acid solution is added, and the solution stirred
for 2 hours, and concentrated. Compound (80) is purified by silica
gel column chromatography.
[0351] Synthesis of Fmoc di-n-butylglycine (81): 10 mmol of
compound (80) is dissolved in 30 mL of dioxane, and 10 mL of 6N
hydrochloric acid solution is added, and the solution is refluxed
overnight. The reaction is cooled to room temperature, concentrated
to dryness, redissolved in 30 mL of tetrahydrofuran, and 10 mL of
water and 30 mmol of sodium bicarbonate is added, followed by 15
mmol of Fmoc-Cl. The biphasic solution is stirred for 1 hour, and
the tetrahydrofuran removed under vacuum. The aqueous solution is
extracted with 1.times.50 mL of diethyl ether, acidified with 1N
hydrochloric acid solution, and extracted with 2.times.50 mL of
ethyl acetate. The ethyl acetate layers are combined, dry over
sodium sulfate, and concentrated. Compound (81) is purified by
silica gel column chromatography.
[0352] Similar methods may be employed by starting with any
appropriate amino acid derivative (similar to compound 78), and by
using an appropriate alkyl butyl, aryl butyl, or aralkyl butyl
reagent the scheme will yield a variety of disubstituted (R, R')
amino acid surrogates where R and R' are different.
##STR00099##
[0353] Synthesis of Fmoc--.alpha.,.alpha. di-n-butyl glycine (87):
To a suspension of 20 mmol of glycine methyl ester hydrochloride
(82), and 2 g of powdered molecular sieves in 40 mL of dry
tetrahydrofuran, kept at room temperature, is added 24 mmol of
potassium hydroxide, followed by 22 mmol of benzaldehyde. The
suspension is stirred for 2 hours, filtered, and the filtrate
concentrated. The residue is redissolved in 40 mL of dry toluene,
and then added to a suspension of 60 mmol of sodium hydride in
toluene, followed by the addition of 60 mmol of n-butyl bromide.
The suspension is stirred for 12 hours, followed by addition of 30
mL of a solution of 6N hydrochloric acid, stirred at room
temperature for 2 hours, and then the layers separated. The
hydrochloride salt of (84) thus obtained is used in situ for
preparation of (87). To isolate (84) as the hydrochloride salt the
aqueous layer is concentrated to dryness and the product
crystallized from dry methanol-ether.
[0354] Alternatively, compound (84) can be prepared from norleucine
methyl ester hydrochloride using a similar synthetic procedure
except that 30 mmol of sodium hydride and 30 mmol of n-butyl
bromide are used for conversion of (86) to (84).
[0355] The aqueous mixture of the hydrochloride form of compound
(84) as obtained above is heated to reflux for 1 hour and then
cooled to room temperature. It is neutralized with solid sodium
hydroxide and then diluted with 30 mL of tetrahydrofuran. Sodium
bicarbonate (30 mmol) is added followed by 15 mmol of Fmoc-Cl. The
biphasic solution is stirred for 1 hour, and the tetrahydrofuran
removed under vacuum. The aqueous solution is extracted with
1.times.50 mL of diethyl ether, acidified with 1N hydrochloric acid
solution, and extracted with 2.times.50 mL of ethyl acetate. The
ethyl acetate layers are combined, dried over sodium sulfate, and
concentrated. Compound (87) is purified by silica gel column
chromatography.
[0356] Similar methods may be employed by starting with any
appropriate amino acid derivative (similar to compound 85), and by
using an appropriate alkyl butyl, aryl butyl, or aralkyl butyl
reagent the scheme will yield a variety of disubstituted (R, R')
amino acid surrogates where R and R' are different.
Synthesis of Disubstituted (R, R') Scaffolds (Method R)
[0357] The invention further provides for constructs in which amino
acid surrogates are employed with two R groups, R and R'. The
following method describes synthesis of Fmoc protected
(R)-5,5-dibutyl-6-oxo-piperazine-2-carboxylic acid, where R and R'
are each groups corresponding to a norleucine side chain moiety. It
may be seen that the method below may be modified, based in part on
the foregoing methods, to produce similar disubstituted (R, R')
amino acid surrogates. Similar methods may be employed such that
starting with any appropriate amino acid derivative (a compound
similar to compound (84)) the scheme can yield a variety of
disubstituted (R, R') amino acid surrogates where R and R' are
different.
##STR00100##
[0358] Synthesis of
(2-Fmoc-amino-3-tert-butoxy-propylamino)-2,2,di-n-butyl acetic acid
methyl ester (88): A suspension of 21 mmol of (84, scheme Q), and
2.9 mL (21 mmol) of triethyl amine in 50 mL of dry tetrahydrofuran,
is stirred at room temperature for 45 minutes, and then a solution
of .about.20 mmol crude Fmoc-(O-t-butyl)-serinal (9, scheme D) in
30 mL of tetrahydrofuran is added, followed by 1.7 g of 4 .ANG.
powdered molecular sieves, and the suspension is stirred for an
additional 2 hours. 6.4 g (30 mmol) of solid sodium
triacetoxyborohydride is added, and the suspension stirred at room
temperature overnight. The suspension is diluted with methanol, the
molecular sieves filtered, and the filtrate concentrated. The
residue is partitioned between 100 mL of ethyl acetate and 50 mL of
water. The organic layer is dried over sodium sulfate, filtered,
and concentrated. Compound (88) is purified by silica gel column
chromatography.
[0359] Synthesis of
4-Fmoc-6-hydroxymethyl-3,3-di-n-butyl-piperazin-2-one (89): A
solution of 10 mmol of compound (88) in 30 mL of 30% diethyl amine
in ethyl acetate is stirred at room temperature overnight, and then
concentrated to dryness. The residue is dissolved in 20 mL of
tetrahydrofuran and 10 mL of water, 2.52 g (30 mmol) of solid
sodium bicarbonate is added, followed by 3.36 g (13 mmol) of
Fmoc-Cl. The mixture is stirred for 3 hours, diluted with 50 mL of
ethyl acetate, the layers separated, and the organic layer washed
with 30 mL of water, dried over magnesium sulfate, and
concentrated. The crude mixture is dissolved in a solution of 10 mL
of 90% trifluoroacetic acid in dichloromethane, stirred for 2
hours, and then concentrated to dryness. The residue is dissolved
in ethyl acetate and washed with 50 mL of a saturated solution of
sodium bicarbonate, dried over magnesium sulfate, and concentrated.
Compound (89) is purified by silica gel column chromatography.
[0360] Synthesis of
4-Fmoc-5,5-di-n-butyl-6-oxo-piperazine-2-carboxylic acid (90): To a
solution of 8 mmol alcohol (89) in 81 mL of acetonitrile kept at
room temperature, is added phosphate buffer solution (prepared with
0.72 g of sodium phosphate monobasic and 1.43 g of sodium phosphate
dibasic in 29.5 mL of water), followed by the addition of 0.33 g
(2.1 mmol) of TEMPO, and 1.86 g (16.5 mmol) of sodium chlorite, and
the biphasic solution is placed in an oil bath kept at 43.degree.
C. A solution of 4.3 mL (2.6 mmol) of sodium hypochlorite solution
(prepared by mixing 1.9 mL of 10-13% sodium hypochlorite solution,
and 2.4 mL of water) is added slowly. The reaction is stirred at
43.degree. C. for 4 hours, cooled to room temperature, 20 mL of 10%
sodium hydrogen sulfite added, stirred for 10 minutes, diluted with
50 mL of ethyl acetate, and the layers separated. The organic layer
is washed with 1.times.10 mL of brine, 1.times.10 mL of 1N
hydrochloric acid solution, dried over sodium sulfate, and
concentrated. Compound (90) is purified by silica gel column
chromatography.
Synthesis of Constructs of the Invention
[0361] The constructs as disclosed in the several embodiments of
this invention may be readily synthesized by any known conventional
procedure for the formation of a peptide linkage between amino
acids. Such conventional procedures include, for example, any
solution phase procedure permitting a condensation between the free
alpha amino group of an amino acid residue having its carboxyl
group or other reactive groups protected and the free primary
carboxyl group of another amino acid residue having its amino group
or other reactive groups protected. In a preferred conventional
procedure, the constructs of this invention may be synthesized by
solid-phase synthesis and purified according to methods known in
the art. The amino acid surrogates of the present invention may be
incorporated into constructs of this invention by methods
substantially similar to or identical to those employed with
residues. Any of a number of well-known procedures utilizing a
variety of resins and reagents may be used to prepare the
constructs of this invention.
[0362] The process for synthesizing the constructs may be carried
out by a procedure whereby each amino acid or amino acid surrogate
in the desired sequence is added one at a time in succession to
another amino acid residue or amino acid surrogate or by a
procedure whereby peptide fragments with the desired amino acid
sequence, which may include one or more amino acid surrogates, are
first synthesized conventionally and then condensed to provide the
desired construct. The resulting construct is cyclized to yield a
cyclic construct of the invention.
[0363] Solid phase peptide synthesis methods are well known and
practiced in the art. In such methods the synthesis of constructs
of the invention can be carried out by sequentially incorporating
the desired amino acid residues or amino acid surrogates one at a
time into the growing peptide chain according to the general
principles of solid phase methods. These methods are disclosed in
numerous references, including Merrifield R. B., Solid phase
synthesis (Nobel lecture). Angew. Chem. 24:799-810 (1985) and
Barany et al., The Peptides, Analysis, Synthesis and Biology, Vol.
2, Gross E. and Meienhofer J., Eds. Academic Press, 1-284
(1980).
[0364] In chemical syntheses of constructs, reactive side chain
groups of the various amino acid residues or amino acid surrogates
are protected with suitable protecting groups, which prevent a
chemical reaction from occurring at that site until the protecting
group is removed. Also common is the protection of the alpha amino
group of an amino acid residue or amino acid surrogate while that
entity reacts at the carboxyl group, followed by the selective
removal of the alpha amino protecting group to allow a subsequent
reaction to take place at that site. Specific protecting groups
have been disclosed and are known in solid phase synthesis methods
and solution phase synthesis methods.
[0365] Alpha amino groups may be protected by a suitable protecting
group, including a urethane-type protecting group, such as
benzyloxycarbonyl (Z) and substituted benzyloxycarbonyl, such as
p-chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl,
p-bromobenzyloxycarbonyl, p-biphenyl-isopropoxycarbonyl,
9-fluorenylmethoxycarbonyl (Fmoc) and p-methoxybenzyloxycarbonyl
(Moz); aliphatic urethane-type protecting groups, such as
t-butyloxycarbonyl (Boc), diisopropylmethoxycarbonyl,
isopropoxycarbonyl, and allyloxycarbonyl. Fmoc is preferred for
alpha amino protection.
[0366] Guanidino groups may be protected by a suitable protecting
group, such as nitro, p-toluenesulfonyl (Tos), Z,
pentamethylchromanesulfonyl (Pmc), adamantyloxycarbonyl,
pentamethyldihydrobenzofuran-5-sulfonyl (Pbf), Fmoc and Boc. Pbf is
one preferred protecting group for Arg. Other preferred protecting
groups include Z, Fmoc, and Boc. It is to be understood that
particularly guanidino protecting groups may be cleaved and removed
during the synthetic process, or may alternatively not be cleaved
or removed, in which event the side chain with the protecting group
forms a derivative of an amino acid side chain moiety as defined
herein. Particularly where the protecting group is labile, and may
be removed by some mechanism in vivo upon administration to a
patient, the construct becomes a "prodrug", which is to say a
construct that is a drug precursor which, following administration
to a patient, is converted to the desired drug form in vivo via
some chemical or physiological process (e.g., a prodrug on being
brought to physiological pH or through enzyme action is converted
to the desired drug form).
[0367] The constructs of the invention described herein can be
prepared using solid phase synthesis, either manually or by means
of an automated peptide synthesizer, using programming modules as
provided by the manufacturer and following the protocols set forth
by the manufacturer, or by modifications of the manufacturers's
protocols to improve the yield of difficult couplings.
[0368] Solid phase synthesis is commenced from the C-terminal end
of the construct by coupling a protected .alpha.-amino acid,
.alpha.-amino acid surrogate or .alpha.-amino alcohol mimetic to a
suitable resin. Such starting material is prepared by attaching an
.alpha.-amino-protected amino acid or .alpha.-amino-protected amino
acid surrogate by an ester linkage to a p-benzyloxybenzyl alcohol
(Wang) resin or a 2-chlorotrityl chloride resin, by an amide bond
between an Fmoc-Linker, such as p-[(R,
S)-.alpha.-[1-(9H-fluor-en-9-yl)-methoxyformamido]-2,4-dimethyloxybenzyl]-
-phenoxyacetic acid (Rink linker) to a benzhydrylamine (BHA) resin,
or by other means well known in the art, such as by attaching an
.alpha.-amino-protected alcohol mimetic to
3,4-dihydro-2H-pyran-2yl-methanol linker attached to chloromethyl
polystyrene resin. Fmoc-Linker-BHA resin supports are commercially
available and generally used when feasible. The resins are carried
through repetitive cycles as necessary to add amino acids
sequentially. The alpha amino Fmoc protecting groups are removed
under basic conditions. Piperidine, piperazine, diethylamine, or
morpholine (20-40% v/v) in N,N-dimethylformamide (DMF) may be used
for this purpose.
[0369] Following removal of the alpha amino protecting group, the
subsequent protected amino acids or amino acid surrogates are
coupled stepwise in the desired order to obtain an intermediate,
protected peptide-resin. The activating reagents used for coupling
of the amino acids in the solid phase synthesis of the peptides are
well known in the art. After the construct is synthesized, if
desired, the orthogonally protected side chain protecting groups
may be removed using methods well known in the art for further
derivatization of the construct.
[0370] Reactive groups in a construct can be selectively modified,
either during solid phase synthesis or after removal from the
resin. For example, constructs can be modified to obtain N-terminus
modifications, such as acetylation, while on resin, or may be
removed from the resin by use of a cleaving reagent and then
modified. Methods for N-terminus modification, such as acetylation,
or C-terminus modification, such as amidation or introduction of an
N-acetyl group, are known in the art. Similarly, methods for
modifying side chains of amino acids are well known to those
skilled in the art of peptide synthesis. The choice of
modifications made to reactive groups present on the construct will
be determined, in part, by the characteristics that are desired in
the construct.
[0371] The construct are, in one embodiment, cyclized prior to
cleavage from the resin. For cyclization through reactive side
chain moieties, the desired side chains are deprotected, and the
construct suspended in a suitable solvent and a cyclic coupling
agent added. Suitable solvents include, for example DMF,
dichloromethane (DCM) or 1-methyl-2-pyrrolidone (NMP). Suitable
cyclic coupling reagents include, for example,
2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
tetrafluoroborate (TBTU),
2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HBTU),
benzotriazole-1-yl-oxy-tris(dimethylamino)phosphoniumhexafluorophosphate
(BOP),
benzotriazole-1-yl-oxy-tris(pyrrolidino)phosphoniumhexafluorophosp-
hate (PyBOP),
2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
tetrafluoroborate (TATU),
2-(2-oxo-1(2H)-pyridyl)-1,1,3,3-tetramethyluronium
tetrafluoroborate (TPTU) or N
N'-dicyclohexylcarbodiimide/1-hydroxybenzotriazole (DCCl/HOBt).
Coupling is conventionally initiated by use of a suitable base,
such as N,N-diispropylethylamine (DIPEA), sym-collidine or
N-methylmorpholine (NMM).
[0372] Following cleavage of constructs from the solid phase
following synthesis, the construct can be purified by any number of
methods, such as reverse phase high performance liquid
chromatography (RP-HPLC), using a suitable column, such as a
C.sub.18 column. Other methods of separation or purification, such
as methods based on the size or charge of the construct, can also
be employed. Once purified, the construct can be characterized by
any number of methods, such as high performance liquid
chromatograph (HPLC), amino acid analysis, mass spectrometry, and
the like.
[0373] Constructs of the present invention with a substituted amide
derivative C-terminus, typically an N-alkyl group, are prepared by
solid phase synthesis commenced from the C-terminal end of the
construct by coupling a protected alpha amino acid or amino acid
surrogate to a suitable resin. Such methods for preparing
substituted amide derivatives on solid phase have been described in
the art. See, for example, Barn D. R., Morphy J. R., Rees D. C.
Synthesis of an array of amides by aluminum chloride assisted
cleavage of resin-bound esters. Tetrahedron Lett. 37, 3213-3216
(1996); DeGrado W. F. Kaiser E. T. Solid-phase synthesis of
protected peptides on a polymer bound oxime: Preparation of
segments comprising the sequences of a cytotoxic 26-peptide
analogue. J. Org. Chem. 47:3258-3261 (1982). Such starting material
can be prepared by attaching an alpha amino-protected amino acid or
amino acid surrogate by an ester linkage to a p-benzyloxybenzyl
alcohol (Wang) resin by well known means. The peptide chain is
grown with the desired sequence of amino acids or amino acid
surrogates, the product cyclized and resin-treated with a solution
of appropriate amine and aluminum chloride (such as methyl amine,
dimethyl amine, ethylamine, and so on) in dichloromethane. The
resulting amide derivative construct is released in solution from
the resin. The resin is filtered and the amide derivative construct
recovered by concentration of solvent followed by precipitation
with ether. The crude construct is dried and remaining amino acid
side chain protective groups cleaved using trifluoroacetic acid
(TFA) in the presence of water and triisopropylsilane (TIS). The
final product is precipitated by adding cold ether and collected by
filtration. Final purification is by RP-HPLC using a C.sub.18
column.
[0374] In one preferred method, the constructs of Example 1 were
synthesized by the following methods. Each of the constructs had
one or two amino acid surrogates based on a keto-piperazine
structure. The amino acid surrogates were synthesized as described
above. The constructs were synthesized using Fmoc chemistry. A
manual synthetic approach was used for couplings immediately before
and after incorporation of the keto-piperazine amino acid
surrogate.
[0375] The following protocol was employed to attach an amino acid
surrogate to resin, such as where the amino acid surrogate was in a
terminal position. Rink amide resin (loading at 0.3 mmol/g,
Advanced ChemTech) was allowed to swell in DMF for 30 minutes. Fmoc
deprotection of the resin was accomplished using 20% piperidine/DMF
for 20 minutes. Coupling of the resin with the selected
Fmoc-protected keto-piperazine amino acid surrogate (2 eq) was
accomplished by overnight incubation in DMF with PyBop (2 eq) and
DIEA (4 eq). If following Kaiser testing a positive result was
obtained, the coupling reaction was conducting a second time.
Acetylation was carried out using Ac.sub.2O (10 eq) and pyridine
(20 eq) in DMF.
[0376] The following protocol was employed to attach a
keto-piperazine amino acid surrogate to peptide-resin. Coupling was
carried out by mixing Fmoc-protected keto piperzine amino acid
surrogate (2 eq), TBTU (2 eq) and DIEA (4 eq) in DMF and allowing
to incubate overnight, again with a repeat of the coupling reaction
if a positive Kaiser test obtained. Acetylation was carried out
using Ac.sub.2O (10 eq) and pyridine (20 eq) in DMF.
[0377] The following protocol was employed to couple an
Fmoc-protected amino acid to a keto-piperazine amino acid surrogate
on solid phase. In most instances at least two coupling cycles were
needed, and frequently three cycles were employed. In a typical
cycle Fmoc-protected amino acid (4 eq) was mixed with HOAt (4 eq)
and DIC (4 eq) in DMF. The resulting mixture was then mixed
overnight in a SPE tube with a keto-piperazine amino acid surrogate
attached directly or through intermediates to resin.
[0378] Couplings between amino acids that were not directly
adjacent to a keto-piperazine amino acid surrogate in the sequence
were conducted using standard protocols for solid phase peptide
synthesis. The following protecting groups were employed: Boc for
Lys and Orn, t-Butyl for Tyr and Ser, Trityl for Cys and His,
O-t-Butyl for Asp and Pbf for Arg.
[0379] Constructs were cleaved from resin employing a mixture of
TFA/thioanisole/phenol/H.sub.2O/DTT/TIS (87.5/2.5/2.5/5/2.5/11) (5
mL) for 3 hours. The resulting material was filtered and
precipitated from cold ether under freezing conditions for one
hour. Precipitated cysteinyl peptide was washed with cold ether at
least three times before being use in an oxidation step.
[0380] For cyclization to form disulfide bonds via air oxidation,
crude cysteinyl construct was dissolved in a mixture of
acetonitrile and water. The pH of the reaction mixture was adjusted
to 7-8 using 5% NH.sub.4OH. The resulted solution was stirred
slowly with 150 mg granular activated carbon for 2 days. Completion
of cyclization was confirmed by LC-MS analysis before proceding to
the next process step. After cyclization, solid carbon was filtered
from solution. The filtrate was lyophilized or dried in a speed-vac
to obtain crude cyclic construct.
[0381] Certain constructs of the invention, where the surrogate is
bound to resin or other peptide solid support and is at the
C-terminal position, may be synthesized by means of the following
scheme. The following scheme is exemplified by synthesis of
construct 1-132, but it is to be understood that substantially
similar methods may be employed for any construct wherein the
surrogate is bound to resin or other peptide solid support.
##STR00101##
[0382] Surrogate (7) is prepared by the scheme of method A above,
or any alternative method. Fmoc protected Sieber amide resin was
treated by swelling 23.8 g (0.63 mmol/g substitution, 15 mmol) of
the resin in 200 mL of a 1:1 mixture of dimethylformamide and
dichloromethane for 45 minutes, followed by filtering and washing
with 2.times.125 mL of dimethylformamide. The washed resin was then
deprotected with 2.times.125 mL of 20% piperidine in
dimethylformamide for 15 minutes, filtered, and washed with
4.times.125 mL of dimethylformamide.
[0383] A solution of 21.5 g (MW=717, 30 mmol) of Fmoc-protected
surrogate (7) in 160 mL of dimethylformamide was added to the
deprotected Sieber amide resin as prepared above, followed by 15.6
g (MW=520.3, 30 mmol) of solid PyBop, and 10.4 mL (MW=129.25,
d=0.742, 60 mmol) of diisopropylethylamine, followed by another 40
mL of dimethylformamide. The mixture was agitated overnight with
nitrogen bubbling. The resin was filtered, and washed with
4.times.130 mL of dimethylformamide, capped with 150 mL of capping
solution consisting of a 3:2:1 solution of dimethylformamide:acetic
anhydride:pyridine for 30 minutes, filtered, and washed with
4.times.130 mL of dimethylformamide to provide surrogate (7)
complexed to resin.
[0384] The resulting Fmoc-protected surrogate (7) complexed to
resin was deprotected with 2.times.130 mL of 20% piperidine in
dimethylformamide for 15 minutes, filtered, and washed with
4.times.130 mL of dimethylformamide to yield surrogate (7)
complexed to resin. A solution of 27.6 g of Fmoc-Tyr-(tBu)-OH (60
mmol, 4 eq.) in dimethylformamide (200 mL) was added to surrogate
(7) complexed to resin, followed by a solution of 24.8 g of HCTU
(60 mmol, 4 eq.), and 20.8 mL (120 mmol, 8 eq.) of
diisopropylethylamine in DMF to a final volume of 200 mL and
coupled overnight with nitrogen bubbling. The resulting
Fmoc-Tyr-(tBu)-surrogate (7)-resin was isolated by filtration and
washed with 2.times.130 mL of dimethylformamide. In order to ensure
complete coupling, the product was again treated with a solution of
27.6 g of Fmoc-Tyr-(tBu)-OH (MW=459.6, 60 mmol, 4 eq.) in
dimethylformamide to a final volume of 200 mL followed by a
solution of 24.8 g of HCTU (60 mmol, 4 eq.), and
diisopropylethylamine (20.8 mL, 120 mmol, 8 eq.) in DMF to a final
volume of 200 mL and coupled overnight with nitrogen bubbling. The
resin was filtered, and washed with 2.times.130 mL of
dimethylformamide. HPLC and LC/MS showed that coupling between
surrogate (7)-resin and Fmoc-Tyr-(tBu)-OH was complete.
[0385] The resulting Fmoc-Tyr-(tBu)-surrogate (7)-resin was then
capped with 150 mL of capping solution as above for 30 minutes. The
resin was then filtered, washed with 4.times.130 mL of
dimethylformamide, 4.times.130 mL of dichloromethane, 2.times.130
mL of MeOH, 2.times.130 mL of diethyl ether, and dried under vacuum
to give 36.7 g.
[0386] Thereafter each succeeding amino acid may be coupled. Before
the coupling of the first amino acid, resulting
Fmoc-Tyr-(tBu)-surrogate (7)-resin was swollen for 45 minutes with
200 mL of a 1:1 solution of dimethylformamide:dichloromethane. Each
amino acid (Fmoc-AA-OH) was coupled by repeating the following
cycle. The terminal amino acid residue was deprotected with
2.times.125 mL of 20% piperidine in dimethylformamide for 15
minutes, filtered and washed with 4.times.125 mL of
dimethylformamide. The beads were checked by ninhydrin test. A
solution of Fmoc-AA-OH (60 mmol, 4 eq.) in dimethylformamide to a
final volume of 200 mL was added to resin, followed by a solution
of HBTU (60 mmol, 4 eq.), and (120 mmol, 8 eq.) of
N-methylmorpholine in DMF to a final volume of 200 mL
[concentration of Fmoc-AA-OH=150 mM solution] and coupled for 30
minutes with nitrogen bubbling (coupling reaction checked by
ninhydrin test). When the ninhydrin test was negative, the resin
was filtered, and washed with 4.times.130 mL of
dimethylformamide.
[0387] After all amino acids had been coupled, the resin was washed
with 4.times.130 mL of dichloromethane, 4.times.130 mL of methanol,
4.times.130 mL of diethyl ether, and dried under vacuum to give
product. The weight increase was quantitative.
[0388] 100 mL of cleavage reagent consisting of a 81.5:5:5:5:2.5:1
solution of trifluoroacetic
acid:phenol:thioanisole:water:DDT:triisopropyl silane was added to
32 g (.about.6.4 mmol) of the following linear construct:
##STR00102##
[0389] The suspension was allowed to stand at room temperature for
5 minutes and then filtered. Another 100 mL of cleavage reagent was
added to the resin, allowed to stand for 5 minutes, and filtered.
This process was repeated.
[0390] The resulting resin was then washed with 2.times.40 mL of
trifluoroacetic acid. The filtrates were combined and stirred for
2.5 hours at room temperature, and then concentrated under reduced
pressure to .about.100 mL volume. Cold diethyl ether (1.5 L,
pre-cooled to -20.degree. C.) was added to the filtrate, and then
placed in the freezer (-20.degree. C.) for 1 hour, filtered through
a sintered glass funnel, and the solids washed with 3.times.200 mL
of cold diethyl ether, and then dried under vacuum for 1 hour with
the solids triturated every 15 minutes to make sure solvent was
removed efficiently. The following construct was obtained (15.4 g)
(103% overall crude yield):
##STR00103##
[0391] The above construct (15.4 g, 6.4 mmol) was dissolved in 16 L
of 30% acetonitrile in water. The pH was adjusted to 8.4 using a
solution of 5% ammonium hydroxide. Pulverized activated carbon
(15.4 g) was added, and the suspension stirred overnight. The
carbon was removed by filtration through celite. The celite was
washed 3.times.100 mL 50% acetonitrile in water. The filtrates were
combined, diluted with water to a final concentration of 10%
acetonitrile, and loaded in the column for purification.
Purification of the trifluoroacetate salt of the resulting
construct was performed under the following conditions:
[0392] Column: Luna C.sub.18, 10 .mu., 50.times.33 mm
[0393] Flow: 70 mL/minute
[0394] Solvent A: water containing 0.1% trifluoroacetic acid
[0395] Solvent B: acetonitrile containing 0.1% trifluoroacetic
acid
[0396] Gradient: 5% solvent B for 5 minutes 26% B to 52% B in 30
minutes
[0397] The pure fractions were combined and lyophilized to give the
purified trifluoroacetate salt of the construct. Dowex SBR, LCNG-OH
resin (450 g) was suspended in 2 L of water, and gently stirred for
15 minutes, allowed to stand for 15 minutes, and then decanted. The
procedure was repeated, and then 0.5 L of water added, and the
slurry transferred into a 6.times.60 cm column. The water was
drained, washed with 4 L of water, and ions exchanged with 6.5 L of
20% acetic acid solution. The resin was allowed to stand at room
temperature overnight, and then washed with water until the pH of
the filtrate was .about.4 (8 L of water used). The trifluoroacetate
salt of the above construct (11.1 g), as prepared above, was
dissolved in 80 mL of water, and loaded to the ion exchange resin,
and eluted with water. Fractions containing 79-1 were combined, and
20% acetic acid solution was added to adjust the final
concentration to 5% acetic acid, and then lyophilized. The
following construct 1-132 (10.4 g) was obtained:
##STR00104##
[0398] Similar methods may be employed with any construct where the
surrogate is bound to resin or other peptide solid support and is
at the C-terminal position.
[0399] Optional PEGylation of the peptide constructs of the
invention may be performed in any manner, such as those described
below.
[0400] PEGylation of reactive amine groups, such as lysine or
ornithine side chains, an omega amino aliphatic in position
Aaa.sup.1, or an amine group in J of an amino acid surrogate at
Aaa.sup.15 was accomplished by dissolving 0.005 mmol purified
construct in 2 mL of dimethylsulfoxide, followed by the addition of
55.5 mg (0.011 mmol, 2 eq) of PEG-5K-OSu (5,000 Da MW methoxy-PEG
with a succinimidyl propionate reactive group), with 17.7 .mu.L
(0.13 mmol, 20 eq.) of triethyl amine then added, and the slightly
cloudy solution stirred at room temperature for 3 hours. Excess
PEG-5K-OSu was quenched by the addition of 7 .mu.L (0.111 mmol, 10
eq.) of ethanol amine, and the reaction stirred overnight.
[0401] PEGylation of reactive carboxyl groups, such as Asp or Glu
side chains or a terminal carboxyl at Aaa.sup.15 on either a
residue or surrogate, is accomplished by coupling PEG-NH.sub.2
(PEG-amine), to the construct containing a carboxylate group in the
side chain of Asp or Glu or at the C-terminus. The peptide
construct (0.005 mmol) is dissolved in DMSO (2 mL), followed by the
addition of 55.5 mg (0.011 mmol, 2 eq) of PEG-NH.sub.2 and HOBt
(0.01 mmol). The coupling is started by the addition of 0.0055
mmole of coupling reagent
N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide (EDAC). The
slightly cloudy solution stirred at room temperature overnight. The
PEGylated peptide construct is then purified by HPLC.
[0402] PEGylation of reactive thiol groups, such as Cys or Hcys
side chains or a thiol group in Q of an amino acid surrogate at
Aaa.sup.1, is accomplished by treating the peptide construct in
DMSO with PEG-methyl-maleimide reagent (SunBio, Orinda, Calif.)
overnight. The PEGylated peptide construct is then purified by
HPLC.
[0403] Following PEGylation, the resulting crude mixture was then
purified by HPLC, yielding a PEG derivatized construct including
one or more amino acid surrogates.
In Vitro and In Vivo Test Systems
[0404] Selected constructs were tested in assays to determine
binding and functional status. The following assays were
employed.
[0405] Cell culture. A cDNA clone that encodes for human
natriuratic peptide receptor A (NPRA) was purchased from Bio
S&T Inc. (Montreal, Quebec). The cDNA clone was inserted into
the mammalian expression vector pcDNA3.1 (Invitrogen) and
transfected into HEK-293 cells. Stable clones were selected by
culture of cells in the presence of G418 sulfate. Expression of
NPRA was examined by binding of [.sup.125I]-atrial natriuretic
peptide ([.sup.125I]-ANP) to membrane homogenates prepared from
clonal cell lines. HEK-hNPRA cells were maintained in culture at
37.degree. C. in 5% CO.sub.2 in Dulbecco's Modified Eagle's Medium
(DMEM) supplemented with 10% FBS, G418 sulfate (300 .mu.g/mL)
sodium glutamate (0.29 mg/mL), penicillin (100 units/mL) and
streptromycin (100 ug/mL).
[0406] Competitive binding assay. A competitive inhibition binding
assay was performed using crude membrane homogenates prepared from
HEK-hNPRA cells. To prepare membrane homogenates, cells were rinsed
with phosphate-buffered saline and incubated for 15 minutes at
4.degree. C. in hypotonic lysis buffer (10 mM Tris, pH 7.4+5 mM
EDTA). Cells were transferred from plates to polypropylene tubes
and homogenized. Homogenates were centrifuged at 25,000.times.g for
20 minutes. Pellets were resuspended in buffer consisting of 50 mM
Tris (pH 7.4) and 1 mM EDTA, homogenized and centrifuged at
25,000.times.g for 20 minutes. Pellets were resuspended in buffer
consisting of 100 mM Tris (pH 7.4) and 10 mM MgCl.sub.2 and stored
at -80.degree. C. until needed. On the day of an assay, homogenates
were thawed and homogenized. Binding of [.sup.125I]-ANP was carried
out in buffer containing 25 mM Hepes (pH 7.4), 100 mM NaCl, 2 mM
CaCl.sub.2, 5 mM MgCl.sub.2, 0.1% BSA and 1 mM 1,10-phenanthroline.
Homogenates (1-10 .mu.g protein/well) were incubated with
[.sup.125I]-ANP (25-30 pM) and increasing concentrations of
competing ligands in Millipore filter plates for 120 minutes at
4.degree. C. Assays were stopped by addition of cold wash buffer
(phosphate-buffered saline) followed by filtration using a vacuum
manifold. Bound radioactivity was determined using a gamma counter.
Non-specific binding was defined by binding of [I.sup.125]-hANP to
non-transfected HEK293 membranes. Data were analyzed using GraphPad
Prism.RTM. curve-fitting software.
[0407] General method for determination of EC.sub.50. Functional
evaluation of constructs was performed by measuring the
accumulation of intracellular cGMP in HEK-293 cells that express
recombinant hNPR-A. HEK-NPRA cells were harvested by washing and
centrifugation in Cell Dissociation Buffer (Gibco, Life
Technologies). Pelleted cells were resuspended in Hank's Balanced
Salt Solution (HBSS) containing 10 mM Hepes (pH 7.4), 5 mM
MgCl.sub.2, 200 mM L-glutamine, 1 mM 1,10-phenanthroline and BSA
(0.5 mg/mL). Following centrifugation, cells were resuspended in
the above buffer supplemented with 0.5 mM
3-isobutyl-1-methylxanthine (IBMX). Cells
(.about.2.times.10.sup.5/well) were added to each well of a 96-well
plate and incubated for 15 minutes at 37.degree. C. Following the
pre-incubation period, cells were incubated for an additional 15
minutes in the presence of increasing concentrations of constructs.
The reaction was terminated by lysis of the cells with temperature
shock. The reaction plate was incubated in a dry ice/ethanol bath
for 15 minutes followed by incubation at 90.degree. C. for 10
minutes. Accumulation of cGMP was measured using the cGMP
Flashplate RIA (Perkin-Elmer). Data analysis and EC.sub.50 values
were determined by using nonlinear regression analysis with
GraphPad Prism.RTM. software.
[0408] Determination of mass and nuclear magnetic resonance
analysis. The mass values of PEG-conjugated constructs were
analyzed by MALDI-TOF mass spectrometry (positive ion mode) using
alpha-cyano-4-hydroxycinnamic acid (CHCA) as matrix. Methanol was
used for sample preparation in construct to matrix ratios of 1:10,
1:20 and 1:30. Alternatively other matrices such as, sinapinic acid
(SA) and 2, 5-dihydroxybenzoic acid (DHB), and solvents such
acetonitrile--0.1% aqueous TFA can be used for sample preparation.
Other determinations of mass values were made using a Waters
MicroMass ZQ device utilizing a positive mode. For constructs that
were not PEGylated, mass determinations were compared with
calculated values and expressed in the form of mass weight plus two
divided by two ((M+2)/2), unless otherwise specified.
[0409] Proton NMR data was obtained using a Bruker 300 MHz
spectrometer. The spectra were obtained after dissolving constructs
in a deuteriated solvent such as chloroform, DMSO, or methanol as
appropriate.
[0410] HPLC measurements were made using a Waters Alliance HT with
a YMC Pack Pro C.sub.18 column (4.6.times.50 mm, 3.mu.) eluted at 1
mL/minute in a step-wise procedure. Solvent A (water containing
0.1% trifluoroacetic acid v/v) and solvent B (acetonitrile
containing 0.1% trifluoroacetic acid v/v) were used as mobile
phases. For analysis of keto piperazine intermediates, the column
was equilibrated with 10% B and then B was increased to 90% over a
period of 8 minutes. For analysis of peptides, the column was
equilibrated with 2% B and then B was increased to 90% over a
period of 8 minutes.
[0411] Animal Models--Blood Pressure Transducer Implantation. Rats
are induced to a surgical plane of anesthesia with isoflurane and
maintained on a heating pad. The abdomen is shaved and scrubbed
with 70% alcohol and betadine solution. Using aseptic technique, a
midline abdominal incision is made in order to expose the
descending aorta and vena cava. The contents of the abdomen are
retracted gently using wet sterile gauze and retractors. Based on
the manufacturer's instructions (described in Data Sciences
International's Multiplus TL Series Device Surgical Manual 2000:
pp. 3.1-3.10), the abdominal aorta is carefully dissected from the
surrounding fat and connective tissue and the catheter of the blood
pressure transducer is inserted. The catheter of the transducer is
secured into place using surgical glue and the body of the
transducer stabilized by suturing to the abdominal wall (4-0 silk
suture). Care is taken to ensure that hemostasis is maintained
during the procedure and that blood flow is not compromised (e.g.
aorta will not be occluded for more than 3 minutes at a time).
Transducer placement is verified using the telemetry radio signal.
After transducer placement, the gauze sponges are removed and the
abdominal cavity is flushed with sterile saline. The abdominal
incision is then sutured closed with nonabsorbable sutures (4-0
silk suture) in a simple interrupted pattern. The skin is closed
using absorbable suture (4-0 vicryl). Finally the animal is removed
from the isoflurane and placed in a warm environment while being
monitored until it is fully awake.
[0412] Surgical Induction of Congestive Heart Failure (Volume
Overload). In this procedure, the descending aorta and vena cava
are exposed in the same manner as it is in the implantation
procedure for the telemetry device. Once access to the vessels
between the renal and iliac bifurcation is obtained, a puncture is
made with a 1.8 mm needle (outside diameter) to the descending
aorta. The needle is advanced into the inferior vena cava and
withdrawn. The ventral puncture site in the descending aorta is
sealed with tissue adhesive. The persistence of a shunt between the
aorta and vena cava is confirmed visually by the swelling of the
vena cava and the mixing of the venous and arterial blood. In the
event that a pressure transducer is also implanted, the two
procedures are done concurrently. The general methods described in
Flaim, S. F., W. J. Minteer, S. H. Nellis, and D. P. Clark: Chronic
arteriovenous shunt: evaluation of a model for heart failure in
rat. Am. J. Physiol. 236:H698-H704 (1979) and Garcia, R. and S.
Diebold: Simple, rapid and effective method of producing aortocaval
shunts in the rat. Cardiovasc. Res. 24:430-432 (1990), are
incorporated here by reference.
[0413] Blood Pressure Monitoring. Telemetry signals from the blood
pressure transducers (model TA11PA-C40, Data Sciences
International, St Paul, Minn.) are collected and analyzed using
Dataquest A.R.T. Gold software version 3.0 (Data Sciences
International). Rats were observed at approximately the same time
each day. Each rat, in its home cage, is placed on a receiver in
the observation room and allowed to adjust to the change in
location for 30 minutes. Baseline recordings are taken for 30
minutes just prior to dosing and treatment recordings are taken for
135 minutes immediately following IV dosing and 210 minutes
following SC dosing. The data are compared to the results after
saline dosing in a way similar to methods previously published in
Clemens, L. E., R. G. Almirez, K. A. Baudouin, E. B. Grossbard, and
A. A. Protter: Human brain natriuretic peptide reduces blood
pressure in normotensive and acute norepinephrine-induced
hypertensive rabbits. Am. J. Hypertens. 10:654-661 (1997),
incorporated here by reference.
[0414] Diuresis and Natriuresis. Rats are induced to a surgical
plane of anesthesia with sodium pentobarbital and maintained on a
heating pad. The abdomen is shaved and scrubbed with 70% alcohol
and betadine solution. Using aseptic technique, a midline abdominal
incision is made in order to expose the urinary bladder. A
purse-string suture is introduced to ventral surface of the bladder
and a small incision is made within the suture area. The flared end
of a catheter in inserted into the open in and the purse-string
suture tightened around it to secure it in place. Urine is
collected into preweighed microcentrifuge tubes at various time
intervals before and after dosing in a way similar to methods
published in Abassi, Z. A., J. R. Powell, E. Golomb, and H. R.
Keiser: Renal and systemic effects of urodilatin in rats with
high-output heart failure. Am. J. Physiol. 262:F615-F621 (1992),
incorporated here by reference. Urine volume is measured by
weight.
EXAMPLE 1
[0415] The following constructs were synthesized, using amino acid
surrogates of one or more of the foregoing methods, were purified
and the mass weights determined, with the results as shown
below:
TABLE-US-00028 TABLE 1 Number (M + 2)/2 Structure 1-1 931.9
##STR00105## 1-2 932.0 ##STR00106## 1-3 938.9 ##STR00107## 1-4
932.0 ##STR00108## 1-5 932.9 ##STR00109## 1-6 932.7 ##STR00110##
1-7 932.3 ##STR00111## 1-8 932.0 ##STR00112## 1-9 932.2
##STR00113## 1-10 931.6 ##STR00114## 1-11 931.6 ##STR00115## 1-12
931.6 ##STR00116## 1-13 931.3 ##STR00117## 1-14 931.6 ##STR00118##
1-15 966.4 ##STR00119## 1-16 966.4 ##STR00120## 1-17 966.0
##STR00121## 1-18 966.4 ##STR00122## 1-19 965.7 ##STR00123## 1-20
965.9 ##STR00124## 1-21 965.7 ##STR00125## 1-22 965.6 ##STR00126##
1-23 1000.4 ##STR00127## 1-24 938.6 ##STR00128## 1-25 931.6
##STR00129## 1-26 938.2 ##STR00130## 1-27 962.2 ##STR00131## 1-28
965.9 ##STR00132## 1-29 932.8 ##STR00133## 1-30 927.8 ##STR00134##
1-31 972.8 ##STR00135## 1-32 972.9 ##STR00136## 1-33 923.2
##STR00137## 1-34 944.7 ##STR00138## 1-35 943.7 ##STR00139## 1-36
923.0 ##STR00140## 1-37 944.6 ##STR00141## 1-38 957.7 ##STR00142##
1-39 965.6 ##STR00143## 1-40 965.8 ##STR00144## 1-41 931.3
##STR00145## 1-42 965.4 ##STR00146## 1-43 965.4 ##STR00147## 1-44
965.5 ##STR00148## 1-45 965.3 ##STR00149## 1-46 965.3 ##STR00150##
1-47 951.3 ##STR00151## 1-48 951.2 ##STR00152## 1-49 958.2
##STR00153## 1-50 965.2 ##STR00154## 1-51 965.3 ##STR00155## 1-52
919.6 ##STR00156## 1-53 965.6 ##STR00157## 1-54 944.6 ##STR00158##
1-55 944.6 ##STR00159## 1-56 930.9 ##STR00160## 1-57 924.0
##STR00161## 1-58 931.0 ##STR00162## 1-59 938.1 ##STR00163## 1-60
896.0 ##STR00164## 1-61 924.1 ##STR00165## 1-62 987.7 ##STR00166##
1-63 931.1 ##STR00167## 1-64 931.1 ##STR00168## 1-65 931.2
##STR00169## 1-66 938.0 ##STR00170## 1-67 1000.1 ##STR00171## 1-68
973.1 ##STR00172## 1-69 924.9 ##STR00173## 1-70 853.5 ##STR00174##
1-71 931.2 ##STR00175## 1-72 874.4 ##STR00176## 1-73 944.4
##STR00177## 1-74 952.1 ##STR00178## 1-75 930.9 ##STR00179## 1-76
1007.1 ##STR00180## 1-77 992.4 ##STR00181## 1-78 958.7 ##STR00182##
1-79 965.6 ##STR00183## 1-80 958.8 ##STR00184## 1-81 964.8
##STR00185## 1-82 948.9 ##STR00186## 1-83 972.7 ##STR00187## 1-84
965.9 ##STR00188## 1-85 993.2 ##STR00189## 1-86 1000.9 ##STR00190##
1-87 993.7 ##STR00191## 1-88 966.7 ##STR00192## 1-89 966.9
##STR00193## 1-90 966.6 ##STR00194## 1-91 967.0 ##STR00195## 1-92
966.9 ##STR00196## 1-93 966.7 ##STR00197## 1-94 966.9 ##STR00198##
1-95 966.7 ##STR00199## 1-96 966.7 ##STR00200## 1-97 966.7
##STR00201## 1-98 966.7 ##STR00202## 1-99 966.5 ##STR00203## 1-100
881.8 ##STR00204## 1-101 922.4 ##STR00205## 1-102 938.0
##STR00206## 1-103 937.8 ##STR00207## 1-104 931.1 ##STR00208##
1-105 931.2 ##STR00209## 1-106 930.9 ##STR00210## 1-107 938.5
##STR00211## 1-108 931.0 ##STR00212## 1-109 945.2 ##STR00213##
1-110 938.1 ##STR00214## 1-111 931.5 ##STR00215## 1-112 1929.6 (M +
1) ##STR00216## 1-113 965.2 ##STR00217## 1-114 965.3 ##STR00218##
1-115 1822.3 (M + 1) ##STR00219## 1-116 1849.3 (M + 1) ##STR00220##
1-117 978.7 ##STR00221## 1-118 965.0 ##STR00222## 1-119 937.0
##STR00223## 1-120 960.5 ##STR00224## 1-121 967.0 ##STR00225##
1-122 981.0 ##STR00226##
1-123 953.4 ##STR00227## 1-124 922.8 ##STR00228## 1-125 965.2
##STR00229## 1-126 967.3 ##STR00230## 1-127 967.3 ##STR00231##
1-128 1030.3 ##STR00232## 1-129 995.7 ##STR00233## 1-130 939.1
##STR00234## 1-131 932.0 ##STR00235## 1-132 938.7 ##STR00236##
1-133 974.0 ##STR00237## 1-134 949 ##STR00238## 1-135 966.3
##STR00239## 1-136 1022.6 ##STR00240## 1-137 1015.5 ##STR00241##
1-138 980.7 ##STR00242## 1-139 930.8 ##STR00243## 1-140 923.8
##STR00244## 1-141 902.9 ##STR00245## 1-142 988.1 ##STR00246##
1-143 1023.4 ##STR00247## 1-144 1033.6 ##STR00248## 1-145 1001.5
##STR00249## 1-146 888.9 ##STR00250## 1-147 1001.6 ##STR00251##
1-148 910.5 ##STR00252## 1-149 967.1 ##STR00253## 1-150 966.0
##STR00254## 1-151 973.1 ##STR00255## 1-152 973.1 ##STR00256##
1-153 923.5 ##STR00257## 1-154 938.1 ##STR00258## 1-155 938.8
##STR00259## 1-156 930.9 ##STR00260## 1-157 931.2 ##STR00261##
1-158 931.3 ##STR00262## 1-159 931.3 ##STR00263## 1-160 931.3
##STR00264## 1-161 931.1 ##STR00265## 1-162 931.3 ##STR00266##
1-163 930.5 ##STR00267## 1-164 938.3 ##STR00268## 1-165 930.8
##STR00269## 1-166 945.1 ##STR00270## 1-167 930.9 ##STR00271##
1-168 931.1 ##STR00272## 1-169 938.0 ##STR00273## 1-170 931.1
##STR00274## 1-171 930.4 ##STR00275## 1-172 930.5 ##STR00276##
1-173 937.7 ##STR00277## 1-174 937.2 ##STR00278## 1-175 930.4
##STR00279## 1-176 916.5 ##STR00280## 1-177 916.4 ##STR00281##
1-178 944.6 ##STR00282## 1-179 937.1 ##STR00283## 1-180 944.6
##STR00284## 1-181 930.4 ##STR00285## 1-182 930.3 ##STR00286##
1-183 930.3 ##STR00287## 1-184 930.9 ##STR00288## 1-185 930.4
##STR00289## 1-186 930.4 ##STR00290## 1-187 937.7 ##STR00291##
1-188 938.8 ##STR00292## 1-189 937.3 ##STR00293## 1-190 937.6
##STR00294## 1-191 938.5 ##STR00295## 1-192 938.4 ##STR00296##
1-193 938.9 ##STR00297## 1-194 938.3 ##STR00298## 1-195 931.5
##STR00299## 1-196 931.4 ##STR00300## 1-197 938.7 ##STR00301##
1-198 931.4 ##STR00302## 1-199 938.3 ##STR00303## 1-200 931.3
##STR00304## 1-201 931.6 ##STR00305## 1-202 938.5 ##STR00306##
1-203 938.2 ##STR00307## 1-204 938.4 ##STR00308## 1-205 937.9
##STR00309## 1-206 930.7 ##STR00310## 1-207 930.7 ##STR00311##
1-208 937.7 ##STR00312## 1-209 930.8 ##STR00313## 1-210 930.5
##STR00314## 1-211 930.5 ##STR00315## 1-212 930.8 ##STR00316##
1-213 931.3 ##STR00317## 1-214 944.9 ##STR00318## 1-215 937.9
##STR00319## 1-216 930.6 ##STR00320## 1-217 930.7 ##STR00321##
1-218 930.8 ##STR00322## 1-219 930.8 ##STR00323## 1-220 930.9
##STR00324## 1-221 931.6 ##STR00325## 1-222 937.8 ##STR00326##
1-223 931.1 ##STR00327## 1-224 938.2 ##STR00328## 1-225 937.8
##STR00329## 1-226 945.4 ##STR00330## 1-227 938.5 ##STR00331##
1-228 930.5 ##STR00332## 1-229 930.4 ##STR00333## 1-230 930.5
##STR00334## 1-231 930.9 ##STR00335## 1-232 938.8 ##STR00336##
1-233 930.8 ##STR00337## 1-234 1030.8 ##STR00338## 1-235 1023.3
##STR00339## 1-236 1031.2 ##STR00340## 1-237 938.1 ##STR00341##
1-238 971.8 ##STR00342## 1-239 955.4 ##STR00343## 1-240 917.4
##STR00344## 1-241 937.8 ##STR00345## 1-242 938.5 ##STR00346##
1-243 930.8 ##STR00347## 1-244 930.8 ##STR00348## 1-245 963.3
##STR00349## 1-246 944.8 ##STR00350## 1-247 959.9 ##STR00351##
1-248 964.3 ##STR00352##
EXAMPLE 2
[0416] The following constructs of Table 2 are synthesized, using
amino acid surrogates of one or more of the foregoing methods, are
purified and the mass weights determined:
TABLE-US-00029 TABLE 2 Num- ber Structure 2-1 ##STR00353## 2-2
##STR00354## 2-3 ##STR00355## 2-4 ##STR00356## 2-5 ##STR00357## 2-6
##STR00358## 2-7 ##STR00359## 2-8 ##STR00360## 2-9 ##STR00361##
2-10 ##STR00362## 2-11 ##STR00363## 2-12 ##STR00364## 2-13
##STR00365## 2-14 ##STR00366## 2-15 ##STR00367## 2-16 ##STR00368##
2-17 ##STR00369## 2-18 ##STR00370## 2-19 ##STR00371## 2-20
##STR00372## 2-21 ##STR00373## 2-22 ##STR00374## 2-23 ##STR00375##
2-24 ##STR00376## 2-25 ##STR00377## 2-26 ##STR00378## 2-27
##STR00379##
EXAMPLE 3
[0417] Construct 1-1 (SEQ ID NO:3), with the following structure,
was tested as described above.
##STR00380##
[0418] In receptor binding studies this construct had an average Ki
of 0.3 nM in an assay system in which hANP had a Ki of 0.05 nM and
mini-ANP had a Ki of 0.6 nM. Construct 1-1 had an EC.sub.50 of 2 nM
in an assay system in which hANP had an EC.sub.50 of 0.6 nM and
mini-ANP had an EC.sub.50 of 3.3 nM.
EXAMPLE 4
[0419] Construct 1-9 (SEQ ID NO:14), (SEQ ID NO:15), with the
following structure, was tested as described above.
##STR00381##
[0420] In receptor binding studies this construct had an average Ki
of 0.9 nM in an assay system in which hANP had a Ki of 0.05 nM and
mini-ANP had a Ki of 0.6 nM. Construct 1-9 had an EC.sub.50 of 3.5
nM in an assay system in which hANP had an EC.sub.50 of 0.6 nM and
mini-ANP had an EC.sub.50 of 3.3 nM.
EXAMPLE 5
[0421] Construct 1-8 (SEQ ID NO:13), with the following structure,
was tested as described above.
##STR00382##
[0422] In receptor binding studies this construct had an average Ki
of 0.2 nM in an assay system in which hANP had a Ki of 0.05 nM and
mini-ANP had a Ki of 0.6 nM. Construct 1-8 had an EC.sub.50 of 2 nM
in an assay system in which the construct of FIG. 1 had an
EC.sub.50 of 0.6 nM and mini-ANP had an EC.sub.50 of 3.3 nM.
EXAMPLE 6
[0423] Construct 1-18 (SEQ ID NO:20), with the following structure,
was tested as described above.
##STR00383##
[0424] In receptor binding studies this construct had an average Ki
of 0.027 nM in an assay system in which hANP had a Ki of 0.05 nM
and mini-ANP had a Ki of 0.6 nM. Construct 1-18 had an EC.sub.50 of
0.2 nM in an assay system in which hANP had an EC.sub.50 of 0.6 nM
and mini-ANP had an EC.sub.50 of 3.3 nM.
[0425] Construct 1-18 was stable in both rat and human plasma, with
T.sub.1/2 of .apprxeq.2 hours at 37.degree. C. When administered
IV, the in vivo T.sub.1/2 in rats was .about.20 minutes.
Approximately 25 to 50% of the injected dose was bioavailable in
rats when administered by a subcutaneous route. FIG. 2 depicts the
concentration of construct 1-18 in ng/mL over time in rats, with
the curve for "SC" indicating subcutaneous administration at a dose
of 5 mg/kg, and the curve for "IV" indicating intravenous
administration at a dose of 2 mg/kg.
EXAMPLE 7
[0426] Blood pressure transducers were implanted in rats as
described under the heading "Animal Models--Blood Pressure
Transducer Implantation." Studies were conducted as described under
the heading "Blood Pressure Monitoring." Studies included
determination of changes in systolic blood pressure compared to
saline following administration of constructs of the invention. In
one study, rats were administered construct 1-63 (SEQ ID NO:20) by
an IV route at 0.03 mg/kg body weight (n=4), 0.1 mg/kg (n=7) or 0.3
mg/kg (n=8). The blood pressure was monitored at 5 minutes prior to
IV administration and at 5, 10 and 15 minutes after administration,
and thereafter at 15 minute intervals until 135 minutes post
administration. At all time points for all doses the measured
systolic blood pressure was lower than saline control, with the
decrease in blood pressure ranging from a minimum of about 5% to a
maximum of about 19%, generally in a dose dependent manner, and
with the greatest response seen at 10 to 45 minutes after
administration.
[0427] In a second study, rats were administered construct 1-63
(SEQ ID NO:20) by a subcutaneous route at 0.3 mg/kg body weight
(n=8), 1.0 mg/kg (n=7) or 3.0 mg/kg (n=7). The blood pressure was
monitored at 15 and 5 minutes prior to SC administration and at 5,
10 and 15 minutes after administration, and thereafter at 15 minute
intervals until 210 minutes post administration. At all time points
for all doses the measured systolic blood pressure was lower than
saline control. At 0.3 mg/kg SC, the decrease in systolic pressure
was in the range of 2% at time points after 2 hours post
administration, which was within the error range. However, at all
other time points for 0.3 mg/kg SC, and at all time points for 1.0
and 3.0 mg/kg, the decrease was statistically different and lower
than the saline control. At 3.0 mg/kg a maximum decrease of
approximately 20% to 23% in systolic pressure was seen at 45 to 120
minutes post administration, with a maximum decrease in the same
time period at 1.0 mg/kg of 17% to 19%.
[0428] In a third study study, rats were administered construct
1-18 (SEQ ID NO:20) by an IV route at 0.3 mg/kg of body weight
(n=8). The blood pressure was monitored at 5 minutes prior to IV
administration and at 5, 10 and 15 minutes after administration,
and thereafter at 15 minute intervals until 135 minutes post
administration. At all time points for all doses the measured
systolic blood pressure was lower than saline control, with the
decrease in blood pressure ranging from a minimum of about 5% to a
maximum of about 13%, with the greatest response seen at 15 minutes
after administration.
[0429] In a fourth study, rats were administered construct 1-18
(SEQ ID NO:20) by a subcutaneous route at 0.1 mg/kg body weight
(n=4), 0.3 mg/kg (n=7) or 1.0 mg/kg (n=8). The blood pressure was
monitored at 15 and 5 minutes prior to SC administration and at 5,
10 and 15 minutes after administration, and thereafter at 15 minute
intervals until 225 minutes post administration. At all time points
for all doses the measured systolic blood pressure was lower than
saline control. At 0.1 mg/kg SC, the decrease in systolic pressure
was not statistically relevant after about two and one-half hours.
However, at all other time points for 0.1 mg/kg SC, and at all time
points less than about two and one-half hours for 0.3 and at all
time points for 1.0 mg/kg, the decrease was statistically different
and lower than the saline control. At 1.0 mg/kg a maximum decrease
of approximately 9% to 13% in systolic pressure was seen at 45 to
120 minutes post administration.
EXAMPLE 8
[0430] Total urine output in rats was measured as described under
the heading "Diuresis and Natriuresis." Groups of four animals were
administered constructs 1-18 (SEQ ID NO:20) and 1-63 (SEQ ID NO:44)
by IV routes, with animals receiving 0.03 mg/kg of body weight, 0.1
mg/kg and 0.3 mg/kg of construct 1-18, and 0.1 mg/kg of body
weight, 0.3 mg/kg and 1.0 mg/kg of construct 1-63(SEQ ID NO:44).
Total urine output over 30 minutes postdose was measured, with
results as shown in FIG. 3.
[0431] In a separate study, total urine about was similarly
measured in a group of four animals receiving doses of 0.3, 1.0 and
3.0 mg/kg of body weight of construct 1-63 (SEQ ID NO:44) by a SC
route, with saline used as a control. Total urine output over 45
minutes postdose was measured. Results are as shown in FIG. 4.
EXAMPLE 9
[0432] The pharmacokinetics of selected constructs of the invention
were studied in male Sprague-Dawley rats following intravenous (IV)
or subcutaneous (SC) administration. Pharmacokinetic parameters of
selected constructs in rats were determined and summarized in
Tables 3 and 4.
[0433] Constructs of the invention as indicated in Tables 4 and 5
were prepared as the TFA salt and dissolved in saline at 1 mL/kg
for both IV and SC dosing routes. The IV dose was administered via
a femoral artery cannula at target doses of 0.3 and 2 mg/kg. The SC
dose was administered at target doses of 1 and 5 mg/kg. Animals
were not fasted overnight before dosing. Blood was collected into
containers containing dipotassium EDTA at predetermined intervals
from a previously implanted cannula in the jugular vein. Plasma was
obtained by centrifugation of the blood and stored at -70.degree.
C. until analysis.
[0434] Data analysis utilized a LC-MS/MS system including a Leap
Technologies HTS-PAL autosampler equipped with a 100 .mu.L
injection loop, two Shimadzu Pumps and a Sciex API 4000 mass
spectrometer. Chromatographic separation of the analytes was
achieved on a Luna C.sub.18 column (4.6.times.100 mm; 3.mu.) eluted
at 1 mL/min with a step-wise procedure. Solvent A (water containing
0.1% formic acid v/v) and solvent B (acetonitrile containing 0.1%
formic acid v/v) were used as the mobile phases. Initially, the
column was equilibrated with 5% B and, 2 minutes after sample
injection, B was increased to 60% over a period of 0.5 minutes and
held at this concentration for 1.1 minutes. The composition of B
was increased to 80% in 1.4 minutes and maintained for 0.3 minutes.
The composition of B was returned to 5% in 0.2 minutes. The total
run time was 6 minutes. Mass spectrometric detection of the
analytes was accomplished using the Turbo Ionspray interface
operated in the positive ion mode. Analyte response was measured by
multiple reaction monitoring (MRM) of the transitions of the
protonated precursor ions to the selected product ions.
[0435] Aliquots of plasma (100 .mu.L) were mixed with the internal
standard (IS) and subjected to solid phase extraction using C.sub.8
cartridges in a 96 well format. After preconditioning of the
C.sub.8 cartridges with 1 mL of methanol and 1 mL of 2% ammonium
hydroxide in water, plasma samples were loaded onto the cartridges.
Following washing the cartridges with 2% ammonium hydroxide in 40%
methanol, constructs were eluted from the cartridges with 1 mL of
2% acetic acid in 60% methanol. The eluents were transferred to a
clean plate, evaporated under a stream of N.sub.2 and the residues
were resuspended in 100 .mu.L of 20 mM ammonium acetate and
acetonitrile (6:4, v:v) prior to LC-MS/MS analysis. Calibration
standards (2-1000 ng/mL) were prepared in the same manner by adding
construct at various concentrations and its respective IS to 100
.mu.L of untreated rat plasma. Similarly, quality control samples
were prepared by adding construct and IS to 100 .mu.L of control
plasma at 3 different concentrations (3.5, 75 and 750 ng/mL) of
construct.
[0436] Data were acquired and processed by Sciex Analyst 1.4.1
software. Peak area ratios of construct to IS were plotted as a
function of the nominal concentrations of construct. Linear
regression using a weighting factor of 1/x was used to calculate
the concentration of construct in plasma samples. The lower limit
of quantification (LLOQ) for this assay typically was 2 or 5
ng/mL.
[0437] Pharmacokinetic parameters were calculated by established
non-compartmental method (Win-Nonlin version 2.1; Consulting Inc.,
Palo Alto, Calif.). The area under the plasma concentration versus
time curve (AUC) was determined using linear trapezoidal
interpolation in the ascending slope and logarithmic trapezoidal
interpolation in the descending slope. The portion of the AUC from
the last measurable concentration to infinity was estimated from
the equation C.sub.t/k.sub.el, where C.sub.t represents the last
measurable concentration and k.sub.el is the elimination rate
constant. The latter was determined from the concentration versus
time curve by linear regression at the terminal phase of the
semi-logarithmic plot.
TABLE-US-00030 TABLE 3 Summary of Pharmacokinetic Parameters of
Constructs in SD Rats Following IV Administration PK Parameters
Dose AUC Vdss Cl (mL/ T.sub.1/2 Construct (mg/kg) (nM hr) (L/kg)
min/kg) (hr) 1-18 (SEQ ID NO: 20) 2 263 0.7 72 0.2 1-18 (SEQ ID NO:
20) 0.3 61 0.4 43 0.2 1-63 (SEQ ID NO: 20) 2 67 4.6 290 0.3 1-63
(SEQ ID NO: 20) 0.3 33 5.8 546 0.3 1-55 (SEQ ID NO: 39) 2 828 0.3
21 0.3 1-55 (SEQ ID NO: 39) 0.3 116 0.2 15 0.2 1-54 (SEQ ID NO: 38)
2 277 1.0 66 0.2 1-35 (SEQ ID NO: 31) 2 10 57 1857 0.4 1-39 2 142
2.8 134 0.3 1-42 2 369 0.7 50 0.3 1-45 2 850 0.3 23 0.3 1-28 2 351
1.1 51 0.4 1-69 (SEQ ID NO: 43) 2 113 0.8 168 0.1 1-100 (SEQ ID NO:
29) 2 42 0.8 456 0.1 1-68 2 478 0.5 40 0.3 1-43 2 1247 0.6 14 0.7
1-44 2 1539 0.4 15 0.5 1-51 (SEQ ID NO: 20) 2 190 1.0 94 0.2
TABLE-US-00031 TABLE 4 Pharmacokinetics of Constructs in SD Rats
Following SC Administration PK Parameters in SD Rats Dose AUC Tmax
Cmax T.sub.1/2 Construct (mg/kg) (nM hr) (hr) (nM) (hr) 1-18 (SEQ
ID NO: 20) 5 255 0.2 131 1.6 1-18 (SEQ ID NO: 20) 1 40 0.1 85 0.2
1-55 (SEQ ID NO: 39) 5 232 0.3 250 0.5 1-63 (SEQ ID NO: 20) 1 7 0.1
26 0.2
EXAMPLE 10
[0438] A formulation of 1-132 was made for pharmaceutical use.
1-132 was used in the acetate salt form. The formulation was
dispensed into a 1 mL vial which was stoppered and sealed, with
each vial containing: [0439] 0.1 mg of 1-132 acetate, based on
peptide weight net of acetate [0440] 1.181 mg succinic acid, NF
[0441] 47.0 mannitol, USP [0442] 1N NaOH, USP, as needed to adjust
pH [0443] 1N HCl, USP, as needed to adjust pH [0444] Water for
injection, to 1 mL volume The pH of the final product was adjusted
to pH 4.00.+-.0.05 with 1N NAOH or 1N HCl, as required. The
resulting solution was filtered through a sterile 0.22 micron
filter prior to vialing, and was stored at 5.degree. C. until
used.
[0445] An alternative formulation of 1-132 was made for
pharmaceutical use, similar to the formulation above, but
additionally including between about 0.02 mg and 0.06 mg of
disodium pamoate, such that the resulting solution was a pamoate
suspension.
EXAMPLE 11
[0446] The following constructs were synthesized, using amino acid
surrogates of one or more of the foregoing methods, purified and
conjugated with PEG-5K-OSu and the mass weights determined, with
the results as shown in Table 5 below:
TABLE-US-00032 TABLE 5 Construct (M + 1) Structure 5-1 6391-8332
##STR00384## 5-2 6338-8412 ##STR00385## 5-3 6427-8159 ##STR00386##
5-4 6406-8219 ##STR00387## 5-5 11959- 13514 ##STR00388## 5-6
6602-8279 ##STR00389## 5-7 6506-8580 ##STR00390## 5-8 6319-8387
##STR00391## 5-9 6660-8505 ##STR00392## 5-10 6444-8174 ##STR00393##
5-11 6383-8103 ##STR00394## 5-12 6479-8289 ##STR00395## 5-13
6564-8869 ##STR00396##
EXAMPLE 12
[0447] The following constructs of Table 6 are synthesized, using
amino acid surrogates of one or more of the foregoing methods,
purified and conjugated with PEG-5K-OSu or another reactive PEG,
and the mass weights determined:
TABLE-US-00033 TABLE 6 Con- struct Structure 6-1 ##STR00397## 6-2
##STR00398## 6-3 ##STR00399## 6-4 ##STR00400## 6-5 ##STR00401## 6-6
##STR00402## 6-7 ##STR00403## 6-8 ##STR00404## 6-9 ##STR00405##
6-10 ##STR00406## 6-11 ##STR00407## 6-12 ##STR00408##
EXAMPLE 13
[0448] Construct 5-1 (SEQ ID NO:83), with the following structure,
was tested as described above.
##STR00409##
[0449] In receptor binding studies this construct had an average Ki
of 70 nM in an assay system in which hANP had a Ki of 0.05 nM and
mini-ANP had a Ki of 0.6 nM.
EXAMPLE 14
[0450] Construct 5-9 (SEQ ID NO:20), with the following structure,
was tested as described above.
##STR00410##
[0451] In receptor binding studies this construct had an average Ki
of approximately 2 nM in an assay system in which hANP had a Ki of
0.05 nM and mini-ANP had a Ki of 0.6 nM. Construct 5-9 had an
EC.sub.50 of approximately 9.5 nM in an assay system in which hANP
had an EC.sub.50 of 0.6 nM and mini-ANP had an EC.sub.50 of 3.3
nM.
EXAMPLE 15
[0452] Any of constructs of the invention, including without
limitation constructs 1-1 to 1-248, 2-1 to 2-21, 5-1 to 5-18 and
6-1 to 6-12, is formulated for time-release injection. Any of the
constructs is formulated with a PEG, such as poly(ethylene glycol)
3350, and optionally one or more additional excipients and
preservatives, including but not limited to excipients such as
salts, polysorbate 80, sodium hydroxide or hydrochloric acid to
adjust pH, and the like. Alternatively, any of the constructs is
formulated with a poly(ortho ester), including an auto-catalyzed
poly(ortho ester) with any of a variable percentage of lactic acid
in the polymeric backbone, and optionally one or more additional
excipients. Poly(D,L-lactide-co-glycolide)polymer (PLGA polymer)
may be employed, preferably a PLGA polymer with a hydrophilic end
group.
EXAMPLE 16
[0453] A patient with congestive heart failure, such as acutely
decomponensated congestive heart failure with dyspnea at rest or
with minimal activity, is administered a formulation including one
or more of any of constructs 1-1 to 1-248, 2-1 to 2-21, 5-1 to 5-18
and 6-1 to 6-12, including a formulation such as by any method of
Example 10, by means of subcutaneous injection.
EXAMPLE 17
[0454] A patient with chronic congestive heart failure is
administered a time release injectable formulation of Example 15 by
means of an injection, such as a deep intramuscular injection, for
example, in the gluteal or deltoid muscle.
EXAMPLE 18
[0455] Applicants have also discovered an inverse correlation
between the extent of resistance to digestion by neutral
endopeptidase of a construct according to the invention and its
pharmacokinetic clearance (CL), as shown in Table 7 below. Neutral
endopeptidase ("NEP") is an endogenous enzyme that inactivates and
clears all three human natriuretic peptides. NEP is present within
renal tubular cells and vascular cells. NEP is highly homologous
between mammalian species. The percent identity of NEP between Rat
and Mouse is 98.5%, between Human and Mouse is 93.6%, and between
Human and Rat is 93.7%.
[0456] NEP resistance of various constructs of the invention was
evaluated using the following experimental procedure. All
constructs were diluted in 0.1 M Tris-HCL buffer (pH 7.4) to 100
.mu.M. 40 .mu.L of diluted constructs were added to each tube and
all tubes were kept on ice. 40 .mu.L of diluted NEP, either human
recombinant NEP (R&D Systems, Minneapolis, Minn., catalogue
#1182-ZN) at 8 ng/.mu.L or mouse NEP (R&D Systems, catalog
#1126-ZN) at 2.5 ng/.mu.L, was added to each tube. The tubes were
mixed gently and spun. All tubes were then incubated at 37.degree.
C. for 0, 0.5, 1, 1.5 and 2 hours. At the end of the incubation
time, the reaction was stopped by adding 5 .mu.L of 10% TFA.
Constructs were linearized using TCEP
(Tris[2-carboxyethyl]phosphine. The extent to which NEP was active
against the constructs was then analyzed by HPLC and/or LC/MS. Data
analysis included determining the percent of remaining starting
material, i.e., undigested peptide construct, and the percent and
sequence of each proteolytic fragment.
TABLE-US-00034 TABLE 7 IV Dosing, PK cGMP hNEP Cl AUC AUCnom Cmax %
of Starting Material Compound ID (mL/min/kg) (nM hr) (nM hr) (nM) 1
h 2 h 1-18 (SEQ ID NO: 20) 55 102 119 172 91 82 1-43 14 238 119 242
97 92 1-44 15 18 9 35 93 86 1-53 40 66 33 105 87 78 1-54 (SEQ ID
NO: 38) 66 128 64 220 85 82 1-55 (SEQ ID NO: 39) 21 116 144 170 83
70 1-63 (SEQ ID NO: 20) 203 89 189 197 67 44 1-100 (SEQ ID NO: 29)
456 8 4 30 6 0 1-118 10 11 6 27 96 92 1-130 92 276 138 524 75 56
1-131 21 161 81 422 87 70 1-133 12 169 563 171 96 93
[0457] Accordingly, one embodiment of the invention provides a
construct according to any of the formulas of the invention that
demonstrates at least 80% of starting material remaining after 1
hour under the hNEP resistance assay conditions described. A
related embodiment of the invention provides a construct according
to any of the formulas of the invention that demonstrates at least
90% of starting material remaining after 1 hour under the hNEP
resistance assay conditions described. A further related embodiment
of the invention provides a construct according to any of the
formulas of the invention that demonstrates at least 95% of
starting material remaining after 1 hour under the hNEP resistance
assay conditions described. In a variation of any of these
embodiments, the construct also demonstrates at least 80% of
starting material remaining after 2 hours under the assay
conditions. In a related variation, the construct also demonstrates
at least 90% of starting material remaining after 2 hours under the
assay conditions.
[0458] Another embodiment of the invention provides a construct
according to any of the formulas of the invention that demonstrates
no more than 90% of starting material remaining after 1 hour under
the hNEP resistance assay conditions described. A related
embodiment of the invention provides a construct according to any
of the formulas of the invention that demonstrates no more than 80%
of starting material remaining after 1 hour under the hNEP
resistance assay conditions described. In a variation of either
embodiment, the construct also demonstrates no more than 90% of
starting material remaining after 2 hours under the assay
conditions. In a related variation, the construct also demonstrates
no more than 80% of starting material remaining after 2 hours under
the assay conditions.
[0459] Thus, constructs of the invention may be selected for
pharmokinetic properties based on the extent of NEP resistance they
demonstrate.
[0460] The preceding examples can be repeated with similar success
by substituting the generically or specifically described reactants
and/or operating conditions of this invention for those used in the
preceding examples.
[0461] Although the invention has been described in detail with
particular reference to these preferred embodiments, other
embodiments can achieve the same results. Variations and
modifications of the present invention will be obvious to those
skilled in the art and it is intended to cover all such
modifications and equivalents. The entire disclosures of all
references, applications, patents, and publications cited above
and/or in the attachments, and of the corresponding application(s),
are hereby incorporated by reference.
Sequence CWU 1
1
101115PRTHomo sapiensDOMAIN(1)..(15)ANP derivative 1Met Cys His Phe
Gly Gly Arg Met Asp Arg Ile Ser Cys Tyr Arg1 5 10 15215PRTHomo
sapiensDOMAIN(1)..(15)ANP derivative 2Met Xaa His Phe Gly Gly Arg
Met Asp Arg Ile Ser Xaa Tyr Arg1 5 10 15314PRTHomo
sapiensMISC_FEATUREPortion of derivative of human ANP with
non-amino acid surrogate 3Cys His Phe Gly Gly Arg Xaa Asp Arg Ile
Ser Cys Tyr Arg1 5 10413PRTHomo sapiensMISC_FEATUREPortion of
derivative of human ANP with non-amino acid surrogate 4Xaa Cys His
Phe Gly Gly Arg Xaa Asp Arg Ile Ser Cys1 5 10511PRTHomo
sapiensMISC_FEATUREPortion of derivative of human ANP with
non-amino acid surrogate 5Gly Gly Arg Xaa Asp Arg Ile Ser Cys Tyr
Arg1 5 10610PRTHomo sapiensMISC_FEATUREPortion of derivative of
human ANP with non-amino acid surrogate 6Xaa Cys His Phe Gly Gly
Arg Xaa Asp Arg1 5 1074PRTHomo sapiensMISC_FEATUREPortion of
derivative of human ANP with non-amino acid surrogate 7Ser Cys Tyr
Arg185PRTHomo sapiensMISC_FEATUREPortion of derivative of human ANP
with non-amino acid surrogate 8Xaa Cys His Phe Gly1 599PRTHomo
sapiensMISC_FEATUREPortion of derivative of human ANP with
non-amino acid surrogate 9Arg Xaa Asp Arg Ile Ser Cys Tyr Arg1
5106PRTHomo sapiensMISC_FEATUREPortion of derivative of human ANP
with non-amino acid surrogate 10Xaa Cys His Phe Gly Gly1
5118PRTHomo sapiensMISC_FEATUREPortion of derivative of human ANP
with non-amino acid surrogate 11Xaa Asp Arg Ile Ser Cys Tyr Arg1
51212PRTHomo sapiensMISC_FEATUREPortion of derivative of human ANP
with non-amino acid surrogate 12Phe Gly Gly Arg Xaa Asp Arg Ile Ser
Cys Tyr Arg1 5 101314PRTHomo sapiensMISC_FEATUREPortion of
derivative of human ANP with non-amino acid surrogate 13Xaa Cys His
Phe Gly Gly Arg Xaa Asp Arg Ile Ser Cys Tyr1 5 10144PRTHomo
sapiensMISC_FEATUREPortion of derivative of human ANP with
non-amino acid surrogate 14Xaa Cys His Phe11510PRTHomo
sapiensMISC_FEATUREPortion of derivative of human ANP with
non-amino acid surrogate 15Gly Arg Xaa Asp Arg Ile Ser Cys Tyr Arg1
5 10166PRTHomo sapiensMISC_FEATUREPortion of derivative of human
ANP with non-amino acid surrogate 16Xaa Cys His Phe Gly Gly1
5178PRTHomo sapiensMISC_FEATUREPortion of derivative of human ANP
with non-amino acid surrogate 17Xaa Asp Arg Ile Ser Cys Tyr Arg1
5185PRTHomo sapiensMISC_FEATUREPortion of derivative of human ANP
with non-amino acid surrogate 18Cys His Phe Gly Gly1 51912PRTHomo
sapiensMISC_FEATUREPortion of derivative of human ANP with
non-amino acid surrogate 19Cys His Phe Gly Gly Arg Xaa Asp Arg Ile
Ser Cys1 5 102013PRTHomo sapiensMISC_FEATUREPortion of derivative
of human ANP with non-amino acid surrogate 20Cys His Phe Gly Gly
Arg Xaa Asp Arg Ile Ser Cys Tyr1 5 102110PRTHomo
sapiensMISC_FEATUREPortion of derivative of human ANP with
non-amino acid surrogate 21Gly Gly Arg Xaa Asp Arg Ile Ser Cys Tyr1
5 10228PRTHomo sapiensMISC_FEATUREPortion of derivative of human
ANP with non-amino acid surrogate 22Arg Xaa Asp Arg Ile Ser Cys
Tyr1 5237PRTHomo sapiensMISC_FEATUREPortion of derivative of human
ANP with non-amino acid surrogate 23Xaa Asp Arg Ile Ser Cys Tyr1
5249PRTHomo sapiensMISC_FEATUREPortion of derivative of human ANP
with non-amino acid surrogate 24Gly Arg Xaa Asp Arg Ile Ser Cys
Tyr1 52513PRTHomo sapiensMISC_FEATUREPortion of derivative of human
ANP with non-amino acid surrogate 25Cys Ala Phe Gly Gly Arg Xaa Asp
Arg Ile Ser Cys Tyr1 5 102613PRTHomo sapiensMISC_FEATUREPortion of
derivative of human ANP with non-amino acid surrogate 26Cys His Ala
Gly Gly Arg Xaa Asp Arg Ile Ser Cys Tyr1 5 102713PRTHomo
sapiensMISC_FEATUREPortion of derivative of human ANP with
non-amino acid surrogate 27Cys His Phe Ala Gly Arg Xaa Asp Arg Ile
Ser Cys Tyr1 5 102813PRTHomo sapiensMISC_FEATUREPortion of
derivative of human ANP with non-amino acid surrogate 28Cys His Phe
Gly Ala Arg Xaa Asp Arg Ile Ser Cys Tyr1 5 102913PRTHomo
sapiensMISC_FEATUREPortion of derivative of human ANP with
non-amino acid surrogate 29Cys His Phe Gly Gly Ala Xaa Asp Arg Ile
Ser Cys Tyr1 5 103013PRTHomo sapiensMISC_FEATUREPortion of
derivative of human ANP with non-amino acid surrogate 30Cys His Phe
Gly Gly Arg Ala Asp Arg Ile Ser Cys Tyr1 5 103113PRTHomo
sapiensMISC_FEATUREPortion of derivative of human ANP with
non-amino acid surrogate 31Cys His Phe Gly Gly Arg Xaa Ala Arg Ile
Ser Cys Tyr1 5 103213PRTHomo sapiensMISC_FEATUREPortion of
derivative of human ANP with non-amino acid surrogate 32Cys His Phe
Gly Gly Arg Xaa Asp Ala Ile Ser Cys Tyr1 5 103313PRTHomo
sapiensMISC_FEATUREPortion of derivative of human ANP with
non-amino acid surrogate 33Cys His Phe Gly Gly Arg Xaa Asp Arg Ala
Ser Cys Tyr1 5 103413PRTHomo sapiensMISC_FEATUREPortion of
derivative of human ANP with non-amino acid surrogate 34Cys His Phe
Gly Gly Arg Xaa Asp Arg Ile Ala Cys Tyr1 5 103513PRTHomo
sapiensMISC_FEATUREPortion of derivative of human ANP with
non-amino acid surrogate 35Cys His Phe Gly Gly Lys Xaa Asp Arg Ile
Ser Cys Tyr1 5 103613PRTHomo sapiensMISC_FEATUREPortion of
derivative of human ANP with non-amino acid surrogate 36Cys His Phe
Gly Gly Arg Xaa Asp Lys Ile Ser Cys Tyr1 5 103713PRTHomo
sapiensMISC_FEATUREPortion of derivative of human ANP with
non-amino acid surrogate 37Cys His Phe Gly Gly Arg Xaa Asp Arg Ile
Ser Cys Ala1 5 103813PRTHomo sapiensMISC_FEATUREPortion of
derivative of human ANP with non-amino acid surrogate 38Cys His Phe
Gly Gly Xaa Xaa Asp Arg Ile Ser Cys Tyr1 5 103913PRTHomo
sapiensMISC_FEATUREPortion of derivative of human ANP with
non-amino acid surrogate 39Cys His Phe Gly Gly Arg Xaa Asp Xaa Ile
Ser Cys Tyr1 5 104014PRTHomo sapiensMISC_FEATUREPortion of
derivative of human ANP with non-amino acid surrogate 40Leu Cys His
Phe Gly Gly Arg Xaa Asp Arg Ile Ser Cys Tyr1 5 104114PRTHomo
sapiensMISC_FEATUREPortion of derivative of human ANP with
non-amino acid surrogate 41Val Cys His Phe Gly Gly Arg Xaa Asp Arg
Ile Ser Cys Tyr1 5 104214PRTHomo sapiensMISC_FEATUREPortion of
derivative of human ANP with non-amino acid surrogate 42Ile Cys His
Phe Gly Gly Arg Xaa Asp Arg Ile Ser Cys Tyr1 5 104313PRTHomo
sapiensMISC_FEATUREPortion of derivative of human ANP with
non-amino acid surrogate 43Cys Ala Phe Gly Gly Arg Xaa Asp Arg Ile
Ala Cys Tyr1 5 104411PRTHomo sapiensMISC_FEATUREPortion of
derivative of human ANP with non-amino acid surrogate 44Cys Phe Gly
Gly Arg Xaa Asp Arg Ile Cys Tyr1 5 10459PRTHomo
sapiensMISC_FEATUREPortion of derivative of human ANP with
non-amino acid surrogate 45Xaa Cys His Phe Gly Gly Arg Xaa Asp1
5465PRTHomo sapiensMISC_FEATUREPortion of derivative of human ANP
with non-amino acid surrogate 46Ile Ser Cys Tyr Arg1 54710PRTHomo
sapiensMISC_FEATUREPortion of derivative of human ANP with
non-amino acid surrogate 47Cys His Phe Gly Gly Arg Xaa Asp Arg Ile1
5 104813PRTHomo sapiensMISC_FEATUREPortion of derivative of human
ANP with non-amino acid surrogate 48Cys His Phe Gly Gly Arg Val Asp
Arg Ile Ser Cys Tyr1 5 104913PRTHomo sapiensMISC_FEATUREPortion of
derivative of human ANP with non-amino acid surrogate 49Cys His Phe
Gly Gly Arg Ile Asp Arg Ile Ser Cys Tyr1 5 105013PRTHomo
sapiensMISC_FEATUREPortion of derivative of human ANP with
non-amino acid surrogate 50Cys His Phe Gly Gly Arg Xaa Asp Arg Val
Ser Cys Tyr1 5 105113PRTHomo sapiensMISC_FEATUREPortion of
derivative of human ANP with non-amino acid surrogate 51Cys His Phe
Gly Gly Arg Xaa Asp Arg Leu Ser Cys Tyr1 5 105213PRTHomo
sapiensMISC_FEATUREPortion of derivative of human ANP with
non-amino acid surrogate 52Cys His Leu Gly Gly Arg Xaa Asp Arg Ile
Ser Cys Tyr1 5 105313PRTHomo sapiensMISC_FEATUREPortion of
derivative of human ANP with non-amino acid surrogate 53Cys His Phe
Gly Gly Arg Leu Asp Arg Ile Ser Cys Tyr1 5 10548PRTHomo
sapiensMISC_FEATUREPortion of derivative of human ANP with
non-amino acid surrogate 54Gly Xaa Asp Arg Ile Ser Cys Tyr1
5558PRTHomo sapiensMISC_FEATUREPortion of derivative of human ANP
with non-amino acid surrogate 55Xaa Cys His Phe Gly Gly Arg Xaa1
5566PRTHomo sapiensMISC_FEATUREPortion of derivative of human ANP
with non-amino acid surrogate 56Arg Ile Ser Cys Tyr Arg1
55713PRTHomo sapiensMISC_FEATUREPortion of derivative of human ANP
with non-amino acid surrogate 57Cys Xaa Phe Gly Gly Xaa Xaa Asp Xaa
Ile Ser Cys Tyr1 5 105813PRTHomo sapiensMISC_FEATUREPortion of
derivative of human ANP with non-amino acid surrogate 58Cys Xaa Phe
Gly Gly Xaa Xaa Asp Xaa Ile Xaa Cys Tyr1 5 105913PRTHomo
sapiensMISC_FEATUREPortion of derivative of human ANP with
non-amino acid surrogate 59Cys His Phe Gly Gly Arg Xaa Asp Arg Ile
Xaa Cys Tyr1 5 106013PRTHomo sapiensMISC_FEATUREPortion of
derivative of human ANP with non-amino acid surrogate 60Cys His Phe
Gly Gly Lys Xaa Asp Lys Ile Ser Cys Tyr1 5 106113PRTHomo
sapiensMISC_FEATUREPortion of derivative of human ANP with
non-amino acid surrogate 61Cys Lys Phe Gly Gly Arg Xaa Asp Arg Ile
Ser Cys Tyr1 5 106213PRTHomo sapiensMISC_FEATUREPortion of
derivative of human ANP with non-amino acid surrogate 62Cys Xaa Phe
Gly Gly Arg Xaa Asp Arg Ile Xaa Cys Tyr1 5 106313PRTHomo
sapiensMISC_FEATUREPortion of derivative of human ANP with
non-amino acid surrogate 63Cys Lys Phe Gly Gly Arg Xaa Asp Arg Ile
Lys Cys Tyr1 5 106413PRTHomo sapiensMISC_FEATUREPortion of
derivative of human ANP with non-amino acid surrogate 64Cys Xaa Phe
Gly Gly Arg Xaa Asp Arg Ile Ser Cys Tyr1 5 106513PRTHomo
sapiensMISC_FEATUREPortion of derivative of human ANP with
non-amino acid surrogate 65Cys His Phe Gly Gly Xaa Xaa Asp Xaa Ile
Ser Cys Tyr1 5 106613PRTHomo sapiensMISC_FEATUREPortion of
derivative of human ANP with non-amino acid surrogate 66Cys His Phe
Gly Gly Arg Xaa Asp Arg Ile Ser Cys Lys1 5 106713PRTHomo
sapiensMISC_FEATUREPortion of derivative of human ANP with
non-amino acid surrogate 67Cys His Phe Gly Gly Arg Xaa Asp Arg Ile
Ser Cys Tyr1 5 106814PRTHomo sapiensMISC_FEATUREPortion of
derivative of human ANP with non-amino acid surrogate 68Cys His Phe
Gly Gly Arg Xaa Asp Arg Ile Ser Cys Tyr Ala1 5 106914PRTHomo
sapiensMISC_FEATUREPortion of derivative of human ANP with
non-amino acid surrogate 69Cys His Phe Gly Gly Arg Xaa Asp Arg Ile
Ser Cys Tyr Arg1 5 107015PRTHomo sapiensMISC_FEATUREPortion of
derivative of human ANP with non-amino acid surrogate 70Cys His Phe
Gly Gly Arg Xaa Asp Arg Ile Ser Cys Tyr Ala Arg1 5 10 157113PRTHomo
sapiensMISC_FEATUREPortion of derivative of human ANP with
non-amino acid surrogate 71Cys His Phe Gly Gly Arg Xaa Asp Arg Ile
Ser Cys Phe1 5 107213PRTHomo sapiensMISC_FEATUREPortion of
derivative of human ANP with non-amino acid surrogate 72Cys His Phe
Gly Gly Arg Xaa Asp Arg Ile Ser Cys Tyr1 5 107313PRTHomo
sapiensMISC_FEATUREPortion of derivative of human ANP with
non-amino acid surrogate 73Cys His Phe Gly Gly Arg Xaa Asp Arg Xaa
Ser Cys Tyr1 5 107413PRTHomo sapiensMISC_FEATUREPortion of
derivative of human ANP with non-amino acid surrogate 74Cys His Phe
Gly Gly Arg Xaa Asp Arg Ile Ser Cys Tyr1 5 107513PRTHomo
sapiensMISC_FEATUREPortion of derivative of human ANP with
non-amino acid surrogate 75Cys His Phe Gly Xaa Arg Xaa Asp Arg Ile
Ser Cys Tyr1 5 107613PRTHomo sapiensMISC_FEATUREPortion of
derivative of human ANP with non-amino acid surrogate 76Cys His Phe
Xaa Gly Arg Xaa Asp Arg Ile Ser Cys Tyr1 5 107713PRTHomo
sapiensMISC_FEATUREPortion of derivative of human ANP with
non-amino acid surrogate 77Cys His Phe Gly Gly Arg Xaa Lys Arg Ile
Ser Cys Tyr1 5 107813PRTHomo sapiensMISC_FEATUREPortion of
derivative of human ANP with non-amino acid surrogate 78Cys His Phe
Gly Gly Arg Xaa Xaa Arg Ile Ser Cys Tyr1 5 107913PRTHomo
sapiensMISC_FEATUREPortion of derivative of human ANP with
non-amino acid surrogate 79Cys His Phe Gly Gly Arg Xaa Asp Arg Ile
Ser Cys Tyr1 5 108013PRTHomo sapiensMISC_FEATUREPortion of
derivative of human ANP with non-amino acid surrogate 80Cys His Phe
Gly Gly Arg Xaa Asp Arg Ile Ser Cys Tyr1 5 108113PRTHomo
sapiensMISC_FEATUREPortion of derivative of human ANP with
non-amino acid surrogate 81Cys His Phe Xaa Gly Arg Xaa Asp Arg Ile
Ser Cys Tyr1 5 108213PRTHomo sapiensMISC_FEATUREPortion of
derivative of human ANP with non-amino acid surrogate 82Cys His Phe
Gly Xaa Arg Xaa Asp Arg Ile Ser Cys Tyr1 5 108313PRTHomo
sapiensMISC_FEATUREPortion of pegylated derivative of human ANP
with non-amino acid surrogate 83Cys Xaa Phe Gly Gly Arg Xaa Asp Arg
Ile Ser Cys Tyr1 5 108413PRTHomo sapiensMISC_FEATUREPortion of
pegylated derivative of human ANP with non-amino acid surrogate
84Cys His Phe Gly Gly Lys Xaa Asp Arg Ile Ser Cys Tyr1 5
108513PRTHomo sapiensMISC_FEATUREPortion of pegylated derivative of
human ANP with non-amino acid surrogate 85Cys His Phe Gly Gly Arg
Xaa Lys Arg Ile Ser Cys Tyr1 5 108613PRTHomo
sapiensMISC_FEATUREPortion of pegylated derivative of human ANP
with non-amino acid surrogate 86Cys Lys Phe Gly Gly Arg Xaa Asp Arg
Ile Ser Cys Tyr1 5 108713PRTHomo sapiensMISC_FEATUREPortion of
pegylated derivative of human ANP with non-amino acid surrogate
87Cys His Phe Gly Gly Xaa Xaa Asp Xaa Ile Ser Cys Tyr1 5
10888PRTHomo sapiensMISC_FEATUREPortion of derivative of human ANP
with non-amino acid surrogate 88Cys His Phe Gly Gly Arg Xaa Asp1
5894PRTHomo sapiensMISC_FEATUREPortion of derivative of human ANP
with non-amino acid surrogate 89Ile Ser Cys Tyr19014PRTHomo
sapiensMISC_FEATUREPortion of derivative of human ANP with
non-amino acid surrogate 90Xaa Cys His Phe Gly Gly Arg Xaa Asp Arg
Ile Ser Cys Tyr1 5 109113PRTHomo sapiensMISC_FEATUREPortion of
pegylated derivative of human ANP with non-amino acid surrogate
91Cys Xaa Phe Gly Gly Xaa Xaa Asp Xaa Ile Xaa Cys Tyr1 5
109213PRTHomo sapiensMISC_FEATUREPortion of pegylated derivative of
human ANP with non-amino acid surrogate 92Cys Xaa Phe Gly Gly Xaa
Xaa Asp Xaa Ile Ser Cys Tyr1 5 109313PRTHomo
sapiensMISC_FEATUREPortion of pegylated derivative of human ANP
with non-amino acid surrogate 93Cys Xaa Phe Gly Gly Arg Xaa Asp Arg
Ile Xaa Cys Tyr1 5 109413PRTHomo sapiensMISC_FEATUREPortion of
pegylated derivative of human ANP with non-amino acid surrogate
94Cys Lys Phe Gly Gly Arg Xaa Asp Arg Ile Lys Cys Tyr1 5
109513PRTHomo sapiensMISC_FEATUREPortion of pegylated derivative of
human ANP with non-amino acid surrogate 95Cys His Phe Gly Gly Lys
Xaa Asp Lys Ile Ser Cys Tyr1 5 109613PRTHomo
sapiensMISC_FEATUREPortion of pegylated derivative of human ANP
with non-amino acid surrogate 96Cys His Phe Gly Gly Arg Xaa Asp Lys
Ile Ser Cys Tyr1 5 109713PRTHomo
sapiensMISC_FEATUREPortion of pegylated derivative of human ANP
with non-amino acid surrogate 97Cys Lys Phe Gly Gly Arg Xaa Asp Lys
Ile Ser Cys Tyr1 5 109813PRTHomo sapiensMISC_FEATUREPortion of
pegylated derivative of human ANP with non-amino acid surrogate
98Cys Xaa Phe Gly Gly Arg Xaa Asp Xaa Ile Ser Cys Tyr1 5
109913PRTHomo sapiensMISC_FEATUREPortion of pegylated derivative of
human ANP with non-amino acid surrogate 99Cys Lys Phe Gly Gly Lys
Xaa Asp Arg Ile Ser Cys Tyr1 5 1010013PRTHomo
sapiensMISC_FEATUREPortion of pegylated derivative of human ANP
with non-amino acid surrogate 100Cys Xaa Phe Gly Gly Xaa Xaa Asp
Arg Ile Ser Cys Tyr1 5 1010113PRTHomo sapiensMISC_FEATUREPortion of
pegylated derivative of human ANP with non-amino acid surrogate
101Cys His Phe Gly Gly Arg Xaa Asp Arg Ile Lys Cys Tyr1 5 10
* * * * *